Protein Detection Methods and Application

Protein Analysis Methods Protein Analysis Methods andand Application Application

LECTURE BY LECTURE BY ANGEL L. SALAMAN-BAYRON, Ph.D.ANGEL L. SALAMAN-BAYRON, Ph.D.

OUTLINEOUTLINE Gel ElectrophoresisGel Electrophoresis Gel Electrophoresis Under denaturing Gel Electrophoresis Under denaturing

conditions conditions Gel Electrophoresis Under non-denaturing Gel Electrophoresis Under non-denaturing

conditions conditions 2D Gel Electrophoresis 2D Gel Electrophoresis Colorimetric AssaysColorimetric Assays Protein ImmunostainingProtein Immunostaining Proteins Colorimetric AssayProteins Colorimetric Assay ElectrofocusingElectrofocusing Non-Traditional (Other) MethodsNon-Traditional (Other) Methods

Gel ElectrophoresisGel Electrophoresis

Gel electrophoresisGel electrophoresis is a technique used for the is a technique used for the separation of deoxyribonucleic acid (DNA), separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix. using an electric current applied to a gel matrix. DNA Gel electrophoresis is generally only used DNA Gel electrophoresis is generally only used after amplification of DNA via PCR. It is usually after amplification of DNA via PCR. It is usually performed for analytical purposes, but may be performed for analytical purposes, but may be used as a preparative technique prior to use of used as a preparative technique prior to use of other methods such as mass spectrometry, other methods such as mass spectrometry, PCR, cloning, DNA sequencing, Southern and PCR, cloning, DNA sequencing, Southern and Western Blotting for further characterization.Western Blotting for further characterization.

Gel ElectrophoresisGel Electrophoresis Proteins, unlike nucleic acids, can have varying charges Proteins, unlike nucleic acids, can have varying charges

and complex shapes, therefore they may not migrate into and complex shapes, therefore they may not migrate into the polyacryl amide gel at similar rates, or at all, when the polyacryl amide gel at similar rates, or at all, when placing a negative to positive EMF on the sample. placing a negative to positive EMF on the sample. Proteins therefore, are usually denatured in the presence Proteins therefore, are usually denatured in the presence of a detergent such as sodium dodecyl sulfate/ of a detergent such as sodium dodecyl sulfate/ (SDS/SDP) that coats the proteins with a negative charge. (SDS/SDP) that coats the proteins with a negative charge. Generally, the amount of SDS bound is relative to the size Generally, the amount of SDS bound is relative to the size of the protein (usually 1.4g SDS per gram of protein), so of the protein (usually 1.4g SDS per gram of protein), so that the resulting denatured proteins have an overall that the resulting denatured proteins have an overall negative charge, and all the proteins have a similar negative charge, and all the proteins have a similar charge to mass ratio. Since denatured proteins act like charge to mass ratio. Since denatured proteins act like long rods instead of having a complex tertiary shape, the long rods instead of having a complex tertiary shape, the rate at which the resulting SDS coated proteins migrate in rate at which the resulting SDS coated proteins migrate in the gel is relative only to its size and not its charge or the gel is relative only to its size and not its charge or shape.shape.

SDS-PAGE (PolyAcrylamide Gel SDS-PAGE (PolyAcrylamide Gel Electrophoresis) Electrophoresis)

The purpose of this method is to separate The purpose of this method is to separate proteins according to their size, and no proteins according to their size, and no other physical feature. In order to other physical feature. In order to understand how this works, we have to understand how this works, we have to understand the two halves of the name: understand the two halves of the name: SDSSDS and and PAGEPAGE SDS: SDS: Sodium Dodecyl Sulfate Sodium Dodecyl Sulfate PAGE:PAGE: Polyacrylamide Gel Electrophoresis Polyacrylamide Gel Electrophoresis

SDS-PAGE (PolyAcrylamide Gel SDS-PAGE (PolyAcrylamide Gel Electrophoresis)Electrophoresis)

SDSSDS (sodium dodecyl sulfate) is a detergent (sodium dodecyl sulfate) is a detergent (soap) that can dissolve hydrophobic molecules (soap) that can dissolve hydrophobic molecules but also has a negative charge (sulfbut also has a negative charge (sulfATEATE) ) attached to it. Therefore, if a cell is incubated attached to it. Therefore, if a cell is incubated with SDS, the membranes will be dissolved and with SDS, the membranes will be dissolved and the proteins will be solubilized by the detergent, the proteins will be solubilized by the detergent, plus all the proteins will be covered with many plus all the proteins will be covered with many negative charges. The end result has two negative charges. The end result has two important features: important features: 1) all proteins contain only primary structure and 1) all proteins contain only primary structure and 2) all proteins have a large negative charge which 2) all proteins have a large negative charge which

means they will all migrate towards the positive pole means they will all migrate towards the positive pole when placed in an electric field when placed in an electric field

SDS-PAGE (PolyAcrylamide Gel SDS-PAGE (PolyAcrylamide Gel Electrophoresis)Electrophoresis)

The top portion of the figure shows The top portion of the figure shows a protein with negative and positive a protein with negative and positive charges due to the charged R-charges due to the charged R-groups of the particular amino acids groups of the particular amino acids in the protein. The large H in the protein. The large H represents hydrophobic domains represents hydrophobic domains where nonpolar R-groups have where nonpolar R-groups have collected in an attempt to get away collected in an attempt to get away from the polar water that surrounds from the polar water that surrounds the protein. The bottom portion the protein. The bottom portion shows that SDS can break up shows that SDS can break up hydrophobic areas and coat hydrophobic areas and coat proteins with many negative proteins with many negative charges which overwhelms any charges which overwhelms any positive charge in the protein due to positive charge in the protein due to positively charged R-groups. The positively charged R-groups. The resulting protein has been resulting protein has been denatured by SDS (reduced to its denatured by SDS (reduced to its primary structure) and as a result primary structure) and as a result has been linearized. has been linearized.

SDS-PAGE (PolyAcrylamide Gel SDS-PAGE (PolyAcrylamide Gel ElectrophoresisElectrophoresis

This Figure shows a slab of polyacrylamide (dark gray) with tunnels (different sized red rings with shading to depict depth) exposed on the edge. Notice that there are many different sizes of tunnels scattered randomly throughout the gel.

Native" or "non-denaturing" gel Native" or "non-denaturing" gel electrophoresis electrophoresis

……is run in the absence of SDS. While in SDS-PAGE the is run in the absence of SDS. While in SDS-PAGE the electrophoretic mobility of proteins depends primarily on their electrophoretic mobility of proteins depends primarily on their molecular mass, in native PAGE the mobility depends on both the molecular mass, in native PAGE the mobility depends on both the protein's charge and its hydrodynamic size.protein's charge and its hydrodynamic size.

The electric charge driving the electrophoresis is governed by the The electric charge driving the electrophoresis is governed by the intrinsic charge on the protein at the pH of the running buffer. This intrinsic charge on the protein at the pH of the running buffer. This charge will, of course, depend on the amino acid composition of the charge will, of course, depend on the amino acid composition of the protein as well as post-translational modifications such as addition protein as well as post-translational modifications such as addition of sialic acids.of sialic acids.

Since the protein retains its folded conformation, its hydrodynamic Since the protein retains its folded conformation, its hydrodynamic size and mobility on the gel will also vary with the nature of this size and mobility on the gel will also vary with the nature of this conformation (higher mobility for more compact conformations, conformation (higher mobility for more compact conformations, lower for larger structures like oligomers). If native PAGE is carried lower for larger structures like oligomers). If native PAGE is carried out near neutral pH to avoid acid or alkaline denaturation, then it out near neutral pH to avoid acid or alkaline denaturation, then it can be used to study conformation, self-association or aggregation, can be used to study conformation, self-association or aggregation, and the binding of other proteins or compounds.and the binding of other proteins or compounds.

Thus native gels can be sensitive to any Thus native gels can be sensitive to any process that alters either the charge or the process that alters either the charge or the conformation of a protein. This makes conformation of a protein. This makes them excellent tools for detecting things them excellent tools for detecting things such as:such as: changes in charge due to chemical degradation (changes in charge due to chemical degradation (e.g. e.g.

deamidation) deamidation) unfolded, "molten globule", or other modified unfolded, "molten globule", or other modified

conformations conformations oligomers and aggregates (both covalent and non-oligomers and aggregates (both covalent and non-

covalent) covalent) binding events (protein-protein or protein-ligand)binding events (protein-protein or protein-ligand)

Native" or "non-denaturing" gel Native" or "non-denaturing" gel electrophoresis electrophoresis

Native" or "non-denaturing" gel Native" or "non-denaturing" gel electrophoresiselectrophoresis

These properties, and their relatively high throughput, These properties, and their relatively high throughput, make native gels excellent tools for analyzing make native gels excellent tools for analyzing accelerated stability samples, demonstrating accelerated stability samples, demonstrating comparability of different lots or processes, or examining comparability of different lots or processes, or examining the effects of excipientsthe effects of excipients..

Another advantage of native gels is that it is possible to Another advantage of native gels is that it is possible to recover proteins in their native state after the separation. recover proteins in their native state after the separation. Recovery of active biological materials may, however, Recovery of active biological materials may, however, need to be done prior to any fixing or staining.need to be done prior to any fixing or staining.

Today many labs unfortunately ignore this valuable tool Today many labs unfortunately ignore this valuable tool because they think native gels are just too hard to use, because they think native gels are just too hard to use, or because they mistakenly believe they can only be or because they mistakenly believe they can only be used with acidic proteins. Bused with acidic proteins. Bothoth basic and acidic proteins, basic and acidic proteins, starting with commercial pre-cast gels and changing the starting with commercial pre-cast gels and changing the buffers can be run. buffers can be run.

Native" or "non-denaturing" gel Native" or "non-denaturing" gel electrophoresiselectrophoresis

It is therefore possible to screen conditions that minimize such oligomer formation using native PAGE. In fact, the figure above shows no dimer formation in histidine, glycine or Tris-HCl buffers (all at 20 mM), consistent with the highly-reversible thermal unfolding of EPO in those conditions.

2D Gel Electrophoresis 2D Gel Electrophoresis

This is a method for the separation and This is a method for the separation and identification of proteins in a sample by identification of proteins in a sample by displacement in 2 dimensions oriented at displacement in 2 dimensions oriented at right angles to one another. This allows right angles to one another. This allows the sample to separate over a larger area, the sample to separate over a larger area, increasing the resolution of each increasing the resolution of each component. A good, general, up to date component. A good, general, up to date reference on this subject is Görg et al, reference on this subject is Görg et al, (2000).(2000).

2D Gel Electrophoresis2D Gel Electrophoresis

How is it Performed?How is it Performed? Isoelectric focusingIsoelectric focusing (IEF) is used in the 1st (IEF) is used in the 1st

Dimension (Righetti, P.G., 1983). This separates Dimension (Righetti, P.G., 1983). This separates proteins by their charge (pI).proteins by their charge (pI).

SDS-PAGE SDS-PAGE in the 2nd Dimension. This in the 2nd Dimension. This separates proteins by their size (molecular separates proteins by their size (molecular weight, MW).weight, MW).

The procedure is known as The procedure is known as ISO-DALTISO-DALT: iso for : iso for isoelectric focusing and dalt for dalton weightisoelectric focusing and dalt for dalton weight

2D Gel Electrophoresis2D Gel Electrophoresis Ispelectrofocusing (IEF)Ispelectrofocusing (IEF) pH gradients can be generated pH gradients can be generated

by adding ampholytes to an acrylamide gel. These are a by adding ampholytes to an acrylamide gel. These are a mixture of amphoteric species with a range of pI values. mixture of amphoteric species with a range of pI values. They are used for carrier ampholyte IEF and in this case They are used for carrier ampholyte IEF and in this case the gel may be pre-focused before sample application.the gel may be pre-focused before sample application.

Ampholyte properties:Ampholyte properties: Even conductivity Even conductivity High buffering capacity High buffering capacity Soluble at isoelectric point Soluble at isoelectric point Minimum interaction with focused proteins Minimum interaction with focused proteins

ImmobilinesImmobilines, s, similar to Ampholytes but have been imilar to Ampholytes but have been immobilised within the polyacrylamide gel producing an immobilised within the polyacrylamide gel producing an immobilised pH gradient or IPG that does not need to be immobilised pH gradient or IPG that does not need to be pre-focused.pre-focused.

2D Gel Electrophoresis2D Gel Electrophoresis ……is generally used as a component of proteomics and is the step is generally used as a component of proteomics and is the step

used for the isolation of proteins for further characterization by mass used for the isolation of proteins for further characterization by mass spectroscopy. Purposes, firstly for the spectroscopy. Purposes, firstly for the large scale identificationlarge scale identification of all of all proteins in a sample. This is undertaken when the global protein proteins in a sample. This is undertaken when the global protein expression of an organism or a tissue is being investigated and is expression of an organism or a tissue is being investigated and is best carried out on model organisms whose genomes have been best carried out on model organisms whose genomes have been fully sequenced. In this way the individual proteins can be more fully sequenced. In this way the individual proteins can be more readily identified from the mass spectrometry data. The second use readily identified from the mass spectrometry data. The second use of this technique is differential expression, this is when you compare of this technique is differential expression, this is when you compare two or more samples to find differences in their two or more samples to find differences in their protein expressionprotein expression. . For instance, you may be looking at drugs resistance in a parasite. For instance, you may be looking at drugs resistance in a parasite. In this case you might like to compare a resistant organism to a In this case you might like to compare a resistant organism to a susceptible one in an attempt to find the changes responsible for the susceptible one in an attempt to find the changes responsible for the resistance. Here the sequence requirements of the organism are not resistance. Here the sequence requirements of the organism are not as important, as you are looking for a relatively small number of as important, as you are looking for a relatively small number of differences and so can devote more time to the identification of each differences and so can devote more time to the identification of each protein. protein.

Proteins Colorimetric AssayProteins Colorimetric Assay Hartree-Lowry and Modified Lowry Protein Assays:Hartree-Lowry and Modified Lowry Protein Assays: The Lowry assay The Lowry assay

(1951) is an often-cited general use protein assay. For some time it (1951) is an often-cited general use protein assay. For some time it was the method of choice for accurate protein determination for cell was the method of choice for accurate protein determination for cell fractions, chromatography fractions, enzyme preparations, and so fractions, chromatography fractions, enzyme preparations, and so on. The bicinchoninic acid (BCA) assay is based on the same on. The bicinchoninic acid (BCA) assay is based on the same principle and can be done in one step, therefore it has been principle and can be done in one step, therefore it has been suggested (Stoscheck, 1990) that the 2-step Lowry method is suggested (Stoscheck, 1990) that the 2-step Lowry method is outdated. However, the modified Lowry is done entirely at room outdated. However, the modified Lowry is done entirely at room temperature. The Hartree version of the Lowry assay, a more recent temperature. The Hartree version of the Lowry assay, a more recent modification that uses fewer reagents, improves the sensitivity with modification that uses fewer reagents, improves the sensitivity with some proteins, is less likely to be incompatible with some salt some proteins, is less likely to be incompatible with some salt solutions, provides a more linear response, and is less likely to solutions, provides a more linear response, and is less likely to become saturated.become saturated.

Reaction Principle:Reaction Principle: Under alkaline conditions the divalent copper ion Under alkaline conditions the divalent copper ion forms a complex with peptide bonds in which it is reduced to a forms a complex with peptide bonds in which it is reduced to a monovalent ion. Monovalent copper ion and the radical groups of monovalent ion. Monovalent copper ion and the radical groups of tyrosine, tryptophan, and cysteine react with Folin reagent to tyrosine, tryptophan, and cysteine react with Folin reagent to produce an unstable product that becomes reduced to produce an unstable product that becomes reduced to molybdenum/tungsten blue molybdenum/tungsten blue

Proteins Colorimetric AssayProteins Colorimetric Assay

Biuret Protein Assay:Biuret Protein Assay: The principle of the Biuret assay The principle of the Biuret assay is similar to that of the Lowry, however it involves a is similar to that of the Lowry, however it involves a single incubation of 20 min. There are very few single incubation of 20 min. There are very few interfering agents (ammonium salts being one such interfering agents (ammonium salts being one such agent), and Layne (1957) reported fewer deviations than agent), and Layne (1957) reported fewer deviations than with the Lowry or ultraviolet absorption methods. with the Lowry or ultraviolet absorption methods. However, the biuret assay consumes much more However, the biuret assay consumes much more material. The biuret is a good general protein assay for material. The biuret is a good general protein assay for batches of material for which yield is not a problem. The batches of material for which yield is not a problem. The Bradford assay is faster and more sensitive.Bradford assay is faster and more sensitive.

Reaction Principle:Reaction Principle: Under alkaline conditions Under alkaline conditions substances containing two or more peptide bonds form a substances containing two or more peptide bonds form a purple complex with copper salts in the reagentpurple complex with copper salts in the reagent

Proteins Colorimetric AssayProteins Colorimetric Assay Bradford protein assay:Bradford protein assay: The Bradford assay is very fast and uses about the The Bradford assay is very fast and uses about the

same amount of protein as the Lowry assay. It is fairly accurate and samples same amount of protein as the Lowry assay. It is fairly accurate and samples that are out of range can be retested within minutes. The Bradford is that are out of range can be retested within minutes. The Bradford is recommended for general use, especially for determining protein content of recommended for general use, especially for determining protein content of cell fractions and assessing protein concentrations for gel electrophoresis. cell fractions and assessing protein concentrations for gel electrophoresis.

Assay materials including color reagent, protein standard, and instruction Assay materials including color reagent, protein standard, and instruction booklet are available from Bio-Rad Corporation. The method described below booklet are available from Bio-Rad Corporation. The method described below is for a 100 µl sample volume using 5 ml color reagent. It is sensitive to about is for a 100 µl sample volume using 5 ml color reagent. It is sensitive to about 5 to 200 micrograms protein, depending on the dye quality. In assays using 5 5 to 200 micrograms protein, depending on the dye quality. In assays using 5 ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µg ml color reagent prepared in lab, the sensitive range is closer to 5 to 100 µg protein. Scale down the volume for the "microassay procedure," which uses 1 protein. Scale down the volume for the "microassay procedure," which uses 1 ml cuvettes. Protocols, including use of microtiter plates are described in the ml cuvettes. Protocols, including use of microtiter plates are described in the flyer that comes with the Bio-Rad kit. flyer that comes with the Bio-Rad kit.

Reaction Principle:Reaction Principle: The assay is based on the observation that the The assay is based on the observation that the absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-absorbance maximum for an acidic solution of Coomassie Brilliant Blue G-250 shifts from 465 nm to 595 nm when binding to protein occurs. Both 250 shifts from 465 nm to 595 nm when binding to protein occurs. Both hydrophobic and ionic interactions stabilize the anionic form of the dye, hydrophobic and ionic interactions stabilize the anionic form of the dye, causing a visible color change. The assay is useful since the extinction causing a visible color change. The assay is useful since the extinction coefficient of a dye-albumin complex solution is constant over a 10-fold coefficient of a dye-albumin complex solution is constant over a 10-fold concentration range.concentration range.

Proteins Colorimetric AssayProteins Colorimetric Assay Bicinchoninic Acid (BCA) Protein Assay (Smith):Bicinchoninic Acid (BCA) Protein Assay (Smith): The The

bicinchoninic acid (BCA) assay is available in kit form from Pierce bicinchoninic acid (BCA) assay is available in kit form from Pierce (Rockford, Ill.). This procedure is very applicable to microtiter plate (Rockford, Ill.). This procedure is very applicable to microtiter plate methods. The BCA is used for the same reasons the Lowry is used. methods. The BCA is used for the same reasons the Lowry is used. Stoscheck (1990) has suggested that the BCA assay will replace Stoscheck (1990) has suggested that the BCA assay will replace the Lowry because it requires a single step, and the color reagent is the Lowry because it requires a single step, and the color reagent is stable under alkaline conditions. stable under alkaline conditions.

Both a standard assay for concentrated proteins and a micro assay for Both a standard assay for concentrated proteins and a micro assay for dilute protein solutions are described below. dilute protein solutions are described below.

Reaction Principle:Reaction Principle: BCA serves the purpose of the Folin reagent in BCA serves the purpose of the Folin reagent in the Lowry assay, namely to react with complexes between copper the Lowry assay, namely to react with complexes between copper ions and peptide bonds to produce a purple end product. The ions and peptide bonds to produce a purple end product. The advantage of BCA is that the reagent is fairly stable under alkaline advantage of BCA is that the reagent is fairly stable under alkaline conditions, and can be included in the copper solution to allow a one conditions, and can be included in the copper solution to allow a one step procedure. A molybdenum/tungsten blue product is produced step procedure. A molybdenum/tungsten blue product is produced as with the Lowry.as with the Lowry.

Protein ImmunostainingProtein Immunostaining

……is a general term in biochemistry that applies is a general term in biochemistry that applies to any use of an antibody-based method to to any use of an antibody-based method to detect a specific protein in a sample. The term detect a specific protein in a sample. The term immunostaining was originally used to refer to immunostaining was originally used to refer to the immunohistochemical staining of tissue the immunohistochemical staining of tissue sections, as first described by Albert Coons in sections, as first described by Albert Coons in 1941.Now however, immunostaining 1941.Now however, immunostaining encompasses a broad range of techniques used encompasses a broad range of techniques used in histology, cell biology, and molecular biology in histology, cell biology, and molecular biology that utilize antibody-based staining methods. that utilize antibody-based staining methods.


ImmunohistochemistryImmunohistochemistry or IHC staining of tissue sections or IHC staining of tissue sections (or immunocytochemistry, which is the staining of cells), (or immunocytochemistry, which is the staining of cells), is perhaps the most commonly applied immunostaining is perhaps the most commonly applied immunostaining technique. While the first cases of IHC staining used technique. While the first cases of IHC staining used fluorescent dyes, other non-fluorescent methods using fluorescent dyes, other non-fluorescent methods using enzymes such as peroxidase and alkaline phosphatase enzymes such as peroxidase and alkaline phosphatase are now used. These enzymes are capable of catalysing are now used. These enzymes are capable of catalysing reactions that give a colored product that is easily reactions that give a colored product that is easily detectable by light microscopy. Alternatively, radioactive detectable by light microscopy. Alternatively, radioactive elements can be used as labels, and the elements can be used as labels, and the immunoreaction can be visualized by autoradiography.immunoreaction can be visualized by autoradiography.

Protein ImmunostainingProtein Immunostaining Tissue preparation or Tissue preparation or fixationfixation is essential for the preservation of cell is essential for the preservation of cell

morphology and tissue architecture. morphology and tissue architecture. Inappropriate or prolonged fixation may significantly diminish the antibody Inappropriate or prolonged fixation may significantly diminish the antibody

binding capability. binding capability. Many antigens can be successfully demonstrated in formalin-fixed Many antigens can be successfully demonstrated in formalin-fixed

paraffin-embedded tissue sections. However, some antigens will not paraffin-embedded tissue sections. However, some antigens will not survive even moderate amounts of aldehyde fixation. Under these survive even moderate amounts of aldehyde fixation. Under these conditions, tissues should be rapidly fresh frozen in liquid nitrogen conditions, tissues should be rapidly fresh frozen in liquid nitrogen and cut with a cryostat. and cut with a cryostat.

The disadvantages of frozen sections include poor morphology, poor The disadvantages of frozen sections include poor morphology, poor resolution at higher magnifications, difficulty in cutting over paraffin resolution at higher magnifications, difficulty in cutting over paraffin sections, and the need for frozen storage. sections, and the need for frozen storage.

Alternatively, vibratome sections do not require the tissue to be Alternatively, vibratome sections do not require the tissue to be processed through organic solvents or high heat, which can destroy processed through organic solvents or high heat, which can destroy the antigenicity, or disrupted by freeze thawing. the antigenicity, or disrupted by freeze thawing.

The disadvantage of vibratome sections is that the sectioning process is The disadvantage of vibratome sections is that the sectioning process is slow and difficult with soft and poorly fixed tissues, and that chatter marks slow and difficult with soft and poorly fixed tissues, and that chatter marks or vibratome lines are often apparent in the sections. or vibratome lines are often apparent in the sections.

Protein ImmunostainingProtein Immunostaining The detection of many antigens can be dramatically improved by The detection of many antigens can be dramatically improved by

antigen retrievalantigen retrieval methods that act by breaking some of the protein methods that act by breaking some of the protein cross-links formed by fixation to uncover hidden antigenic sites. This cross-links formed by fixation to uncover hidden antigenic sites. This can be accomplished by heating for varying lengths of times (heat can be accomplished by heating for varying lengths of times (heat induced epitope retrieval or HIER) or using enzyme digestion induced epitope retrieval or HIER) or using enzyme digestion (proteolytic induced epitope retrieval or PIER).(proteolytic induced epitope retrieval or PIER).

One of the main difficulties with IHC staining is overcoming specific One of the main difficulties with IHC staining is overcoming specific or non-specific background. Optimisation of fixation methods and or non-specific background. Optimisation of fixation methods and times, pre-treatment with blocking agents, incubating antibodies with times, pre-treatment with blocking agents, incubating antibodies with high salt, and optimising post-antibody wash buffers and wash times high salt, and optimising post-antibody wash buffers and wash times are all important for obtaining high quality immunostaining. In are all important for obtaining high quality immunostaining. In addition, the presence of positive and negative controls for staining addition, the presence of positive and negative controls for staining are essential for determining specificity.are essential for determining specificity.

Protein ImmunostainingProtein Immunostaining A A flow cytometerflow cytometer is a technology that allows a single cell to be is a technology that allows a single cell to be

measured for a variety of characteristics, determined by looking at measured for a variety of characteristics, determined by looking at how they flow in liquid. Instruments used for this can gather how they flow in liquid. Instruments used for this can gather information about cells by measuring visible and fluorescent light information about cells by measuring visible and fluorescent light emissions, allowing cell sorting based on physical, biochemical and emissions, allowing cell sorting based on physical, biochemical and antigenic traits.antigenic traits.

It can be used for the direct analysis of cells expressing one or more It can be used for the direct analysis of cells expressing one or more specific proteins. Cells are immunostained in solution using specific proteins. Cells are immunostained in solution using methods similar to used for immunofluorescence, and then analyzed methods similar to used for immunofluorescence, and then analyzed by flow cytometry.by flow cytometry.

Flow cytometry has several advantages over IHC including: the Flow cytometry has several advantages over IHC including: the ability to define distinct cell populations are defined by their size and ability to define distinct cell populations are defined by their size and granularity; the capacity to gate out dead cells; improved sensitivity; granularity; the capacity to gate out dead cells; improved sensitivity; and multi-colour analysis to measure several antigens and multi-colour analysis to measure several antigens simultaneously. However, flow cytometry can be less effective at simultaneously. However, flow cytometry can be less effective at detecting extremely rare cell populations, and there is a loss of detecting extremely rare cell populations, and there is a loss of architectural relationships in the absence of a tissue section. architectural relationships in the absence of a tissue section.

Flow cytometerFlow cytometer

Protein ImmunostainingProtein Immunostaining Western blottingWestern blotting allows the detection of specific proteins allows the detection of specific proteins

from extracts made from cells or tissues, before or after from extracts made from cells or tissues, before or after any purification steps. Proteins are generally separated any purification steps. Proteins are generally separated by size using gel electrophoresis before being transferred by size using gel electrophoresis before being transferred to a synthetic membrane via dry, semi-dry, or wet blotting to a synthetic membrane via dry, semi-dry, or wet blotting methods. The membrane can then be probed using methods. The membrane can then be probed using antibodies using methods similar to antibodies using methods similar to immunohistochemistry, but without a need for fixation. immunohistochemistry, but without a need for fixation. Detection is typically performed using peroxidase linked Detection is typically performed using peroxidase linked antibodies to catalyse a chemiluminescent reaction.antibodies to catalyse a chemiluminescent reaction.

Western blotting is a routine molecular biology method Western blotting is a routine molecular biology method that can be used to semi-quantitatively compare protein that can be used to semi-quantitatively compare protein levels between extracts. The size separation prior to levels between extracts. The size separation prior to blotting allows the protein molecular weight to be gauged blotting allows the protein molecular weight to be gauged as compared with known molecular weight markersas compared with known molecular weight markers

No bands present Neg

Bands at either p31 OR p24 AND bands present at either gp160 OR gp120

Pos

Bands present, but pattern does not meet criteria for positivity

+/-

Western BlottingWestern Blotting

Western BlottingWestern Blotting Western blotting transfer Western blotting transfer

apparatusapparatus.. Schematic showing Schematic showing the assembly of a typical the assembly of a typical Western blot apparatus with the Western blot apparatus with the position of the position of the position of the position of the gel, transfer membrane and gel, transfer membrane and direction of protein in relation to direction of protein in relation to the electrode position. Although the electrode position. Although the image depicted here is the image depicted here is representative of a vertical "wet" representative of a vertical "wet" transfer apparatus, the transfer apparatus, the orientation is applicable for orientation is applicable for horizontally positioned semi-dry horizontally positioned semi-dry transfer apparatus. transfer apparatus.

Western BlottingWestern Blotting


The enzyme-linked immunosorbent assay or ELISA is a The enzyme-linked immunosorbent assay or ELISA is a diagnostic method for quantitatively or semi-diagnostic method for quantitatively or semi-quantitatively determining protein concentrations from quantitatively determining protein concentrations from blood plasma, serum or cell/tissue extracts in a multi-well blood plasma, serum or cell/tissue extracts in a multi-well plate format (usually 96-wells per plate). Broadly, plate format (usually 96-wells per plate). Broadly, proteins in solution are adsorbed to ELISA plates. proteins in solution are adsorbed to ELISA plates. Antibodies specific for the protein of interest are used to Antibodies specific for the protein of interest are used to probe the plate. Background is minimized by optimizing probe the plate. Background is minimized by optimizing blocking and washing methods (as for IHC), and blocking and washing methods (as for IHC), and specificity is ensured via the presence of positive and specificity is ensured via the presence of positive and negative controls. Detection methods are usually negative controls. Detection methods are usually colorimetric or chemiluminescence based. colorimetric or chemiluminescence based.


Advantages •Quicker since only one antibody is used •No concern for cross-reactivity of a secondary antibody •Double possible with different labels on primary antibodies Disadvantages •Labeling may reduce immunoreactivity of primary antibody •Labeled primary antibodies are expensive •Low flexibility in choice of primary antibody label •Little signal amplification

Advantages •Secondary antibody can amplify signal •A variety of labeled secondary antibodies are available •One secondary may be used with many primary antibodies •Labeling does not affect primary antibody immunoreactivity •Changing secondary allows change of detection methodDisadvantages •Secondary antibodies may produce nonspecific staining •Additional steps required compared to the direct method

ELISA ASSAY PROCESS

Electron microscopyElectron microscopy

……or EM can be used to study the detailed or EM can be used to study the detailed microarchitecture of tissues or cells. Immuno-EM microarchitecture of tissues or cells. Immuno-EM allows the detection of specific proteins in allows the detection of specific proteins in ultrathin tissue sections. Antibodies labelled with ultrathin tissue sections. Antibodies labelled with heavy metal particles (e.g. gold) can be directly heavy metal particles (e.g. gold) can be directly visualised using transmission electron visualised using transmission electron microscopy. While powerful in detecting the sub-microscopy. While powerful in detecting the sub-cellular localization of a protein, immuno-EM can cellular localization of a protein, immuno-EM can be technically challenging, expensive, and be technically challenging, expensive, and require rigorous optimization of tissue fixation require rigorous optimization of tissue fixation and processing methods. and processing methods.

Protein Assays by Protein Assays by Spectrophotometry Spectrophotometry

PrinciplesPrinciples A spectrophotometer consists of two instruments, namely a A spectrophotometer consists of two instruments, namely a

spectrometerspectrometer for producing light of any selected color (wavelength), for producing light of any selected color (wavelength), and a and a photometerphotometer for measuring the intensity of light. The for measuring the intensity of light. The instruments are arranged so that liquid in a cuvette can be placed instruments are arranged so that liquid in a cuvette can be placed between the spectrometer beam and the photometer. The amount between the spectrometer beam and the photometer. The amount of light passing through the tube is measured by the photometer. of light passing through the tube is measured by the photometer. The photometer delivers a voltage signal to a display device, The photometer delivers a voltage signal to a display device, normally a galvanometer. The signal changes as the amount of light normally a galvanometer. The signal changes as the amount of light absorbed by the liquid changes.absorbed by the liquid changes.

If development of color is linked to the concentration of a substance If development of color is linked to the concentration of a substance in solution then that concentration can be measured by determining in solution then that concentration can be measured by determining the extent of absorption of light at the appropriate wavelength. For the extent of absorption of light at the appropriate wavelength. For example hemoglobin appears red because the hemoglobin absorbs example hemoglobin appears red because the hemoglobin absorbs blue and green light rays much more effectively than red. The blue and green light rays much more effectively than red. The degree of absorbance of blue or green light is proportional to the degree of absorbance of blue or green light is proportional to the concentration of hemoglobin.concentration of hemoglobin.

Protein Assays by Protein Assays by SpectrophotometrySpectrophotometry

Principles (cont.)Principles (cont.) When monochromatic light (light of a specific wavelength) passes through a solution When monochromatic light (light of a specific wavelength) passes through a solution

there is usually a quantitative relationship (Beer's law) between the solute there is usually a quantitative relationship (Beer's law) between the solute concentration and the intensity of the transmitted light, that is, concentration and the intensity of the transmitted light, that is,

I = II = Ioo X 10 X 10--kc1kc1

where where II sub 0 is the intensity of transmitted light using the pure solvent, sub 0 is the intensity of transmitted light using the pure solvent, II is the is the intensity of the transmitted light when the colored compound is added, c is intensity of the transmitted light when the colored compound is added, c is concentration of the colored compound, concentration of the colored compound, ll is the distance the light passes through the is the distance the light passes through the solution, and k is a constant. If the light path solution, and k is a constant. If the light path ll is a constant, as is the case with a is a constant, as is the case with a spectrophotometer, Beer's law may be written, spectrophotometer, Beer's law may be written,

I / II / Ioo = 10 = 10--kckc = T = T where where k k is a new constant and is a new constant and TT is the transmittance of the solution. There is a is the transmittance of the solution. There is a

logarithmic relationship between transmittance and the concentration of the colored logarithmic relationship between transmittance and the concentration of the colored compound. Thus, compound. Thus,

-log T-log T = = log 1/Tlog 1/T = = kckc = Optical Density (OD) = Optical Density (OD) The O.D. is directly proportional to the concentration of the colored compound. Most The O.D. is directly proportional to the concentration of the colored compound. Most

spectrophotometers have a scale that reads both in O.D. (absorbance) units, which is spectrophotometers have a scale that reads both in O.D. (absorbance) units, which is a logarithmic scale, and in % transmittance, which is an arithmetic scale. As a logarithmic scale, and in % transmittance, which is an arithmetic scale. As suggested by the above relationships, the absorbance scale is the most useful for suggested by the above relationships, the absorbance scale is the most useful for colorimetric assayscolorimetric assays


Proteins in solution absorb ultraviolet light Proteins in solution absorb ultraviolet light with absorbance maxima @ 280 and 200 with absorbance maxima @ 280 and 200 nm. Amino acids with aromatic rings are nm. Amino acids with aromatic rings are the primary reason for the absorbance the primary reason for the absorbance peak at 280 nm. Peptide bonds are peak at 280 nm. Peptide bonds are primarily responsible for the peak at 200 primarily responsible for the peak at 200 nm. Secondary, tertiary, and quaternary nm. Secondary, tertiary, and quaternary structure all affect absorbance, therefore structure all affect absorbance, therefore factors such as pH, ionic strength, etc. can factors such as pH, ionic strength, etc. can alter the absorbance spectrum.alter the absorbance spectrum.


Unknown proteins or protein mixturesUnknown proteins or protein mixtures.. Use the following Use the following formula to roughly estimate protein concentration. Path formula to roughly estimate protein concentration. Path length for most spectrometers is 1 cm. length for most spectrometers is 1 cm.

ConcentrationConcentration (mg/ml) = Absorbance at 280 nm divided by path (mg/ml) = Absorbance at 280 nm divided by path length (cm.) length (cm.)

Pure protein of known absorbance coefficientPure protein of known absorbance coefficient.. Use the Use the following formula for a path length of 1 cm. Concentration following formula for a path length of 1 cm. Concentration is in mg/ml, %, or molarity depending on which type is in mg/ml, %, or molarity depending on which type coefficient is used. coefficient is used.

ConcentrationConcentration = Absorbance at 280 nm divided by absorbance = Absorbance at 280 nm divided by absorbance coefficient coefficient

To convert units, use these relationships: To convert units, use these relationships: Mg protein/ml = % protein divided by 10 = molarity divided by Mg protein/ml = % protein divided by 10 = molarity divided by

protein molecular weight protein molecular weight Unknowns with possible nucleic acid contaminationUnknowns with possible nucleic acid contamination.. Use Use

the following formula to estimate protein concentration: the following formula to estimate protein concentration: Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260) Concentration (mg/ml) = (1.55 x A280) - 0.76 x A260)

Enzyme AssaysEnzyme Assays ……are laboratory methods for measuring enzymatic activity. They are vital for are laboratory methods for measuring enzymatic activity. They are vital for

the study of enzyme kinetics and enzyme inhibition. the study of enzyme kinetics and enzyme inhibition. Amounts of enzymes can either be expressed as molar amounts, as with any Amounts of enzymes can either be expressed as molar amounts, as with any

other chemical, or measured in terms of activity, in enzyme units. other chemical, or measured in terms of activity, in enzyme units. Enzyme activityEnzyme activity = moles of substrate converted per unit time = rate × = moles of substrate converted per unit time = rate ×

reaction volume. Enzyme activity is a measure of the quantity of active reaction volume. Enzyme activity is a measure of the quantity of active enzyme present and is thus dependent on conditions, enzyme present and is thus dependent on conditions, which should be which should be specifiedspecified. The SI unit is the katal, 1 katal = 1 mol s-1, but this is an . The SI unit is the katal, 1 katal = 1 mol s-1, but this is an excessively large unit. A more practical and commonly-used value is 1 excessively large unit. A more practical and commonly-used value is 1 enzyme unit (EU) = 1 μmol min-1 (μ = micro, x 10-6). 1 U corresponds to enzyme unit (EU) = 1 μmol min-1 (μ = micro, x 10-6). 1 U corresponds to 16.67 nanokatals.16.67 nanokatals.

Specific activitySpecific activity of an enzyme is another common unit. This is the activity of of an enzyme is another common unit. This is the activity of an enzyme per milligram of total protein (expressed in μmol min-1mg-1). an enzyme per milligram of total protein (expressed in μmol min-1mg-1). Specific activity gives a measurement of the purity of the enzyme. It is the Specific activity gives a measurement of the purity of the enzyme. It is the amount of product formed by an enzyme in a given amount of time under amount of product formed by an enzyme in a given amount of time under given conditions per milligram of enzyme. Specific activity is equal to the rate given conditions per milligram of enzyme. Specific activity is equal to the rate of reaction multiplied by the volume of reaction divided by the mass of of reaction multiplied by the volume of reaction divided by the mass of enzyme. The SI unit is katal kg-1, but a more practical unit is μmol mg-1 min-enzyme. The SI unit is katal kg-1, but a more practical unit is μmol mg-1 min-1. Specific activity is a measure of enzyme processivity, usually constant for 1. Specific activity is a measure of enzyme processivity, usually constant for a pure enzyme.a pure enzyme.

Enzyme AssaysEnzyme Assays

Related terminologyRelated terminology The The rate of a reactionrate of a reaction is the concentration of is the concentration of

substrate disappearing (or product produced) substrate disappearing (or product produced) per unit time (per unit time (molmol LL − 1 − 1ss − 1) − 1)

The The % purity% purity is 100% × (specific activity of is 100% × (specific activity of enzyme sample / specific activity of pure enzyme sample / specific activity of pure enzyme). The impure sample has lower specific enzyme). The impure sample has lower specific activity because some of the mass is not actually activity because some of the mass is not actually enzyme. If the specific activity of 100% pure enzyme. If the specific activity of 100% pure enzyme is known, then an impure sample will enzyme is known, then an impure sample will have a lower specific activity, allowing purity to have a lower specific activity, allowing purity to be calculated.be calculated.

Types of Enzyme AssaysTypes of Enzyme Assays All enzyme assays measure either the consumption of substrate or production of All enzyme assays measure either the consumption of substrate or production of

product over time. A large number of different methods of measuring the product over time. A large number of different methods of measuring the concentrations of substrates and products exist and many enzymes can be assayed in concentrations of substrates and products exist and many enzymes can be assayed in several different ways. Biochemists usually study enzyme-catalyzed reactions using several different ways. Biochemists usually study enzyme-catalyzed reactions using four types of experiments:four types of experiments:

Initial rate experimentsInitial rate experiments. When an enzyme is mixed with a large excess of the . When an enzyme is mixed with a large excess of the substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then substrate, the enzyme-substrate intermediate builds up in a fast initial transient. Then the reaction achieves a steady-state kinetics in which enzyme substrate intermediates the reaction achieves a steady-state kinetics in which enzyme substrate intermediates remains approximately constant over time and the reaction rate changes relatively remains approximately constant over time and the reaction rate changes relatively slowly. Rates are measured for a short period after the attainment of the quasi-steady slowly. Rates are measured for a short period after the attainment of the quasi-steady state, typically by monitoring the accumulation of product with time. Because the state, typically by monitoring the accumulation of product with time. Because the measurements are carried out for a very short period and because of the large excess measurements are carried out for a very short period and because of the large excess of substrate, the approximation free substrate is approximately equal to the initial of substrate, the approximation free substrate is approximately equal to the initial substrate can be made. The initial rate experiment is the simplest to perform and substrate can be made. The initial rate experiment is the simplest to perform and analyze, being relatively free from complications such as back-reaction and enzyme analyze, being relatively free from complications such as back-reaction and enzyme degradation. It is therefore by far the most commonly used type of experiment in degradation. It is therefore by far the most commonly used type of experiment in enzyme kinetics.enzyme kinetics.

Progress curve experimentsProgress curve experiments.. In these experiments, the kinetic parameters are In these experiments, the kinetic parameters are determined from expressions for the species concentrations as a function of time. The determined from expressions for the species concentrations as a function of time. The concentration of the substrate or product is recorded in time after the initial fast concentration of the substrate or product is recorded in time after the initial fast transient and for a sufficiently long period to allow the reaction to approach equilibrium. transient and for a sufficiently long period to allow the reaction to approach equilibrium. We note in passing that, while they are less common now, progress curve experiments We note in passing that, while they are less common now, progress curve experiments were widely used in the early period of enzyme kinetics.were widely used in the early period of enzyme kinetics.

Types of Enzyme Assays (cont.)Types of Enzyme Assays (cont.) Transient kinetics experimentsTransient kinetics experiments. In these experiments, reaction behaviour . In these experiments, reaction behaviour

is tracked during the initial fast transient as the intermediate reaches the is tracked during the initial fast transient as the intermediate reaches the steady-state kinetics period. These experiments are more difficult to perform steady-state kinetics period. These experiments are more difficult to perform than either of the above two classes because they require rapid mixing and than either of the above two classes because they require rapid mixing and observation techniques.observation techniques.

Relaxation experimentsRelaxation experiments. In these experiments, an equilibrium mixture of . In these experiments, an equilibrium mixture of enzyme, substrate and product is perturbed, for instance by a temperature, enzyme, substrate and product is perturbed, for instance by a temperature, pressure or pH jump, and the return to equilibrium is monitored. The analysis pressure or pH jump, and the return to equilibrium is monitored. The analysis of these experiments requires consideration of the fully reversible reaction. of these experiments requires consideration of the fully reversible reaction. Moreover, relaxation experiments are relatively insensitive to mechanistic Moreover, relaxation experiments are relatively insensitive to mechanistic details and are thus not typically used for mechanism identification, although details and are thus not typically used for mechanism identification, although they can be under appropriate conditions.they can be under appropriate conditions.

Enzyme assays can be split into two groups according to their sampling Enzyme assays can be split into two groups according to their sampling method: method:

Continues assaysContinues assays where the assay gives a continuous reading of activity, and where the assay gives a continuous reading of activity, and Discontinuous assaysDiscontinuous assays when samples are taken from an enzyme reaction at when samples are taken from an enzyme reaction at

intervals and the amount of product production or substrate consumption is intervals and the amount of product production or substrate consumption is measured in these samples. Continuous assays are most convenient, with one measured in these samples. Continuous assays are most convenient, with one assay giving the rate of reaction with no further work necessary. assay giving the rate of reaction with no further work necessary.

Types of Enzyme Assays (cont.)Types of Enzyme Assays (cont.)

Direct versus coupled assaysDirect versus coupled assays Coupled assay for hexokinase using glucose-6-Coupled assay for hexokinase using glucose-6-

phosphate dehydrogenase.phosphate dehydrogenase. Even when the enzyme reaction does not result in a Even when the enzyme reaction does not result in a

change in the absorbance of light, it can still be possible change in the absorbance of light, it can still be possible to use a spectrophotometric assay for the enzyme by to use a spectrophotometric assay for the enzyme by using a using a coupled assaycoupled assay. Here, the product of one . Here, the product of one reaction is used as the substrate of another, easily-reaction is used as the substrate of another, easily-detectable reaction. For example, figure 1 shows the detectable reaction. For example, figure 1 shows the coupled assay for the enzyme hexokinase, which can be coupled assay for the enzyme hexokinase, which can be assayed by coupling its production of glucose-6-assayed by coupling its production of glucose-6-phosphate to NADPH production, using glucose-6-phosphate to NADPH production, using glucose-6-phosphate dehydrogenase.phosphate dehydrogenase.

Types of Continuous Enzyme Types of Continuous Enzyme AssaysAssays

FluorometricFluorometric ( (Fluorescence) is when a molecule emits Fluorescence) is when a molecule emits light of one wavelength after absorbing light of a different light of one wavelength after absorbing light of a different wavelength. Fluorometric assays use a difference in the wavelength. Fluorometric assays use a difference in the fluorescence of substrate from product to measure the fluorescence of substrate from product to measure the enzyme reaction. These assays are in general much enzyme reaction. These assays are in general much more sensitive than spectrophotometric assays, but can more sensitive than spectrophotometric assays, but can suffer from interference caused by impurities and the suffer from interference caused by impurities and the instability of many fluorescent compounds when exposed instability of many fluorescent compounds when exposed to light.to light.

An example of these assays is again the use of the nucleotide An example of these assays is again the use of the nucleotide coenzymes NADH and NADPH. Here, the reduced forms are coenzymes NADH and NADPH. Here, the reduced forms are fluorescent and the oxidised forms non-fluorescent. Oxidation fluorescent and the oxidised forms non-fluorescent. Oxidation reactions can therefore be followed by a decrease in reactions can therefore be followed by a decrease in fluorescence and reduction reactions by an increase. Synthetic fluorescence and reduction reactions by an increase. Synthetic substrates that release a fluorescent dye in an enzyme-catalyzed substrates that release a fluorescent dye in an enzyme-catalyzed reaction are also available, such as 4-methylumbelliferyl-β-D-reaction are also available, such as 4-methylumbelliferyl-β-D-galactoside for assaying β-galactosidase.galactoside for assaying β-galactosidase.


CalorimetricCalorimetric ( (Calorimetry) is the measurement of the heat released Calorimetry) is the measurement of the heat released or absorbed by chemical reactions. These assays are very general, or absorbed by chemical reactions. These assays are very general, since many reactions involve some change in heat and with use of a since many reactions involve some change in heat and with use of a microcalorimeter, not much enzyme or substrate is required. These microcalorimeter, not much enzyme or substrate is required. These assays can be used to measure reactions that are impossible to assays can be used to measure reactions that are impossible to assay in any other way.assay in any other way.

ChemiluminescentChemiluminescent ( (Chemiluminescence) is the emission of light Chemiluminescence) is the emission of light by a chemical reaction. Some enzyme reactions produce light and by a chemical reaction. Some enzyme reactions produce light and this can be measured to detect product formation. These types of this can be measured to detect product formation. These types of assay can be extremely sensitive, since the light produced can be assay can be extremely sensitive, since the light produced can be captured by photographic film over days or weeks, but can be hard captured by photographic film over days or weeks, but can be hard to quantify, because not all the light released by a reaction will be to quantify, because not all the light released by a reaction will be detected.detected.

The detection of horseradish peroxidase by enzymatic The detection of horseradish peroxidase by enzymatic chemiluminescence (ECL) is a common method of detecting antibodies chemiluminescence (ECL) is a common method of detecting antibodies in western blotting. Another example is the enzyme luciferase, this is in western blotting. Another example is the enzyme luciferase, this is found in fireflies and naturally produces light from its substrate luciferin.found in fireflies and naturally produces light from its substrate luciferin.


Light ScatteringLight Scattering:: measures the product of measures the product of weight-averaged molar mass and concentration weight-averaged molar mass and concentration of macromolecules in solution. Given a fixed of macromolecules in solution. Given a fixed total concentration of one or more species over total concentration of one or more species over the measurement time, the scattering signal is a the measurement time, the scattering signal is a direct measure of the weight-averaged molar direct measure of the weight-averaged molar mass of the solution, which will vary as mass of the solution, which will vary as complexes form or dissociate. Hence the complexes form or dissociate. Hence the measurement quantifies the stoichiometry of the measurement quantifies the stoichiometry of the complexes as well as kinetics. Light scattering complexes as well as kinetics. Light scattering assays of protein kinetics is a very general assays of protein kinetics is a very general technique that does not require an enzyme.technique that does not require an enzyme.

Types of Discontinuous Enzyme Types of Discontinuous Enzyme AssaysAssays

RadiometricRadiometric assays measure the incorporation of radioactivity into assays measure the incorporation of radioactivity into substrates or its release from substrates. The radioactive isotopes most substrates or its release from substrates. The radioactive isotopes most frequently used in these assays are 14C, 32P, 35S and 125I. Since frequently used in these assays are 14C, 32P, 35S and 125I. Since radioactive isotopes can allow the specific labeling of a single atom of a radioactive isotopes can allow the specific labeling of a single atom of a substrate, these assays are both extremely sensitive and specific. They are substrate, these assays are both extremely sensitive and specific. They are frequently used in biochemistry and are often the only way of measuring a frequently used in biochemistry and are often the only way of measuring a specific reaction in crude extracts (the complex mixtures of enzymes specific reaction in crude extracts (the complex mixtures of enzymes produced when you lyse cells).produced when you lyse cells).

Radioactivity is usually measured in these procedures using a scintillation Radioactivity is usually measured in these procedures using a scintillation counter.counter.

ChromatographicChromatographic assays measure product formation by separating the assays measure product formation by separating the reaction mixture into its components by chromatography. This is usually reaction mixture into its components by chromatography. This is usually done by high-performance liquid chromatography (HPLC), but can also use done by high-performance liquid chromatography (HPLC), but can also use the simpler technique of thin layer chromatography. Although this approach the simpler technique of thin layer chromatography. Although this approach can need a lot of material, its sensitivity can be increased by labeling the can need a lot of material, its sensitivity can be increased by labeling the substrates/products with a radioactive or fluorescent tag. Assay sensitivity substrates/products with a radioactive or fluorescent tag. Assay sensitivity has also been increased by switching protocols to improved has also been increased by switching protocols to improved chromatographic instruments (e.g. ultra-high pressure liquid chromatographic instruments (e.g. ultra-high pressure liquid chromatography) that operate at pump pressure a few-fold higher than chromatography) that operate at pump pressure a few-fold higher than HPLC instrumentsHPLC instruments

Factors to control in Enzyme Factors to control in Enzyme AssaysAssays

Salt Concentration:Salt Concentration: Most enzymes cannot tolerate extremely high salt Most enzymes cannot tolerate extremely high salt concentrations. The ions interfere with the weak ionic bonds of proteins. concentrations. The ions interfere with the weak ionic bonds of proteins. Typical enzymes are active in salt concentrations of 1-500 mM. As usual Typical enzymes are active in salt concentrations of 1-500 mM. As usual there are exceptions such as the halophilic (salt loving) algae and bacteria. there are exceptions such as the halophilic (salt loving) algae and bacteria.

Effects of Temperature:Effects of Temperature: All enzymes work within a range of temperature All enzymes work within a range of temperature specific to the organism. Increases in temperature generally lead to specific to the organism. Increases in temperature generally lead to increases in reaction rates. There is a limit to the increase because higher increases in reaction rates. There is a limit to the increase because higher temperatures lead to a sharp decrease in reaction rates. This is due to the temperatures lead to a sharp decrease in reaction rates. This is due to the denaturating (alteration) of protein structure resulting from the breakdown of denaturating (alteration) of protein structure resulting from the breakdown of the weak ionic and hydrogen bonding that stabilize the three dimensional the weak ionic and hydrogen bonding that stabilize the three dimensional structure of the enzyme. The "optimum" temperature for human enzymes is structure of the enzyme. The "optimum" temperature for human enzymes is usually between 35 and 40 °C. The average temperature for humans is 37 usually between 35 and 40 °C. The average temperature for humans is 37 °C. Human enzymes start to denature quickly at temperatures above 40 °C. °C. Human enzymes start to denature quickly at temperatures above 40 °C.

Enzymes from thermophilic archaea found in the hot springs are stable up to 100 Enzymes from thermophilic archaea found in the hot springs are stable up to 100 °C. However, the idea of an "optimum" rate of an enzyme reaction is misleading, °C. However, the idea of an "optimum" rate of an enzyme reaction is misleading, as the rate observed at any temperature is the product of two rates, the reaction as the rate observed at any temperature is the product of two rates, the reaction rate and the denaturation rate. If you were to use an assay measuring activity for rate and the denaturation rate. If you were to use an assay measuring activity for one second, it would give high activity at high temperatures, however if you were one second, it would give high activity at high temperatures, however if you were to use an assay measuring product formation over an hour, it would give you low to use an assay measuring product formation over an hour, it would give you low activity at these temperatures. activity at these temperatures.

Factors to control in Enzyme Factors to control in Enzyme AssaysAssays

Effects of pH:Effects of pH: Most enzymes are sensitive to pH and have specific Most enzymes are sensitive to pH and have specific ranges of activity. All have an optimum pH. The pH can stop ranges of activity. All have an optimum pH. The pH can stop enzyme activity by denaturating (altering) the three dimensional enzyme activity by denaturating (altering) the three dimensional shape of the enzyme by breaking ionic, and hydrogen bonds. Most shape of the enzyme by breaking ionic, and hydrogen bonds. Most enzymes function between a pH of 6 and 8; however pepsin in the enzymes function between a pH of 6 and 8; however pepsin in the stomach works best at a pH of 2 and trypsin at a pH of 8. stomach works best at a pH of 2 and trypsin at a pH of 8.

Substrate Saturation:Substrate Saturation: Increasing the substrate concentration Increasing the substrate concentration increases the rate of reaction (enzyme activity). However, enzyme increases the rate of reaction (enzyme activity). However, enzyme saturation limits reaction rates. An enzyme is saturated when the saturation limits reaction rates. An enzyme is saturated when the active sites of all the molecules are occupied most of the time. At active sites of all the molecules are occupied most of the time. At the saturation point, the reaction will not speed up, no matter how the saturation point, the reaction will not speed up, no matter how much additional substrate is added. The graph of the reaction rate much additional substrate is added. The graph of the reaction rate will plateau. will plateau.

Level of crowdingLevel of crowding, large amounts of macromolecules in a solution , large amounts of macromolecules in a solution will alter the rates and equilibrium constants of enzyme reactions, will alter the rates and equilibrium constants of enzyme reactions, through an effect called macromolecular crowding.through an effect called macromolecular crowding.

Immune FluorescenceImmune Fluorescence Most commonly, immunofluorescence employs two sets of Most commonly, immunofluorescence employs two sets of

antibodies: a primary antibody is used against the antigen of interest; antibodies: a primary antibody is used against the antigen of interest; a subsequent, secondary ("indirect"), dye-coupled antibody is a subsequent, secondary ("indirect"), dye-coupled antibody is introduced that recognizes the primary antibody. In this fashion the introduced that recognizes the primary antibody. In this fashion the researcher may create several primary antibodies that recognize researcher may create several primary antibodies that recognize various antigens, but, because they all share a common constant various antigens, but, because they all share a common constant region, may be recognized by a single dye-coupled antibody. region, may be recognized by a single dye-coupled antibody. Typically this is done by using antibodies made in different species. Typically this is done by using antibodies made in different species. For example, a researcher might create antibodies in a goat that For example, a researcher might create antibodies in a goat that recognize several antigens, and then employ dye-coupled rabbit recognize several antigens, and then employ dye-coupled rabbit antibodies that recognize the goat antibody constant region (denoted antibodies that recognize the goat antibody constant region (denoted rabbit anti-goat). This allows re-use of the difficult-to-make dye-rabbit anti-goat). This allows re-use of the difficult-to-make dye-coupled antibodies in multiple experiments.coupled antibodies in multiple experiments.

In some cases, it is advantageous to use primary antibodies directly In some cases, it is advantageous to use primary antibodies directly labelled with a fluorophore. This direct labelling decreases the labelled with a fluorophore. This direct labelling decreases the number of steps in the staining procedure and, more importantly, number of steps in the staining procedure and, more importantly, often avoids cross-reactivity and high background problems. often avoids cross-reactivity and high background problems. Fluorescent labelling can be performed in less than one hour with Fluorescent labelling can be performed in less than one hour with readily available labeling kits.readily available labeling kits.

Immune FluorescenceImmune Fluorescence As with most fluorescence techniques, a significant problem with As with most fluorescence techniques, a significant problem with

immunofluorescence is photobleaching. Loss of activity caused by immunofluorescence is photobleaching. Loss of activity caused by photobleaching can be controlled by reducing the intensity or time-photobleaching can be controlled by reducing the intensity or time-span of light exposure, by increasing the concentration of span of light exposure, by increasing the concentration of fluorophores, or by employing more robust fluorophores that are fluorophores, or by employing more robust fluorophores that are less prone to bleaching (e.g. Alexa Fluors or DyLight Fluors).less prone to bleaching (e.g. Alexa Fluors or DyLight Fluors).

Many uses of immunofluorescence have been outmoded by the Many uses of immunofluorescence have been outmoded by the development of recombinant proteins containing fluorescent protein development of recombinant proteins containing fluorescent protein domains, e.g. green fluorescent protein (GFP). Use of such "tagged" domains, e.g. green fluorescent protein (GFP). Use of such "tagged" proteins allows much better localization and less disruption of proteins allows much better localization and less disruption of protein function.protein function.

Method overview Method overview

1.1. In immunostaining methods, an In immunostaining methods, an antibodyantibody is used to is used to detect a specific detect a specific proteinprotein epitopeepitope. These antibodies can . These antibodies can be be monoclonalmonoclonal or or polyclonalpolyclonal. Detection of this first or . Detection of this first or primary antibodyprimary antibody can be accomplished in multiple can be accomplished in multiple ways.ways.

2.2. The primary antibody can be directly labeled using an The primary antibody can be directly labeled using an enzymeenzyme or or fluorophorefluorophore. .

3.3. The primary antibody can be labeled using a small The primary antibody can be labeled using a small molecule which interacts with a high affinity binding molecule which interacts with a high affinity binding partner that can be linked to an enzyme or fluorophore. partner that can be linked to an enzyme or fluorophore. The The biotinbiotin-strepavidin is one commonly used high -strepavidin is one commonly used high affinity interaction. affinity interaction.

Method overviewMethod overview

4.4. The primary antibody can be probed for using a The primary antibody can be probed for using a broader species-specific broader species-specific secondary antibodysecondary antibody that is that is labeled using an enzyme, or fluorophore. labeled using an enzyme, or fluorophore.

5.5. In the case of In the case of electron microscopyelectron microscopy, antibodies are , antibodies are linked to a heavy metal linked to a heavy metal atomatom. .

6.6. As previously described, enzymes such as As previously described, enzymes such as horseradishhorseradish peroxidaseperoxidase or or alkaline alkaline phosphatasephosphatase are commonly are commonly used to catalyse reactions that give a coloured or used to catalyse reactions that give a coloured or chemiluminescentchemiluminescent product. Fluorescent molecules can product. Fluorescent molecules can be visualised using be visualised using fluoresence microscopyfluoresence microscopy or or confocal confocal microscopymicroscopy..

ApplicationsApplications The applications of The applications of immunostainingimmunostaining are are

numerous, but are most typically used in numerous, but are most typically used in clinical clinical diagnosticsdiagnostics and and laboratory researchlaboratory research..

Clinically, IHC is used in Clinically, IHC is used in histopathologyhistopathology for the for the diagnosis of specific types of cancers based on diagnosis of specific types of cancers based on molecular markers.molecular markers.

In laboratory science, immunostaining can be In laboratory science, immunostaining can be used for a variety of applications based on used for a variety of applications based on investigating the presence or absence of a investigating the presence or absence of a protein, its tissue distribution, its sub-cellular protein, its tissue distribution, its sub-cellular localization, and or changes in protein localization, and or changes in protein expression or degradationexpression or degradation

Non-Traditional (Other) MethodsNon-Traditional (Other) Methods

Mass Spectrometry:Mass Spectrometry: A mass spectrometer creates charged particles A mass spectrometer creates charged particles (ions) from molecules. It then analyzes those ions to provide (ions) from molecules. It then analyzes those ions to provide information about the molecular weight of the compound and its information about the molecular weight of the compound and its chemical structure. There are many types of mass spectrometers chemical structure. There are many types of mass spectrometers and sample introduction techniques which allow a wide range of and sample introduction techniques which allow a wide range of analyses. This discussion will focus on mass spectrometry as it's analyses. This discussion will focus on mass spectrometry as it's used in the powerful and widely used method of coupling Gas used in the powerful and widely used method of coupling Gas Chromatography (GC) with Mass Spectrometry (MS). Chromatography (GC) with Mass Spectrometry (MS).

Gas Chromatograph (GC):Gas Chromatograph (GC): A mixture of compounds to be analysed A mixture of compounds to be analysed is initially injected into the GC where the mixture is vaporized in a is initially injected into the GC where the mixture is vaporized in a heated chamber. The gas mixture travels through a GC column, heated chamber. The gas mixture travels through a GC column, where the compounds become separated as they interact with the where the compounds become separated as they interact with the column. The chromatogram on the right shows peaks which result column. The chromatogram on the right shows peaks which result from this separation. Those separated compounds then immediately from this separation. Those separated compounds then immediately enter the mass spectrometer.enter the mass spectrometer.

Mass Spectrometer (MS)Mass Spectrometer (MS)

All mass All mass spectrometers consist spectrometers consist of three distinct of three distinct regions. regions. 1) Ionizer 1) Ionizer

2) Ion analyzer 2) Ion analyzer

3) Detector 3) Detector

Mass Spectrometer (MS)Mass Spectrometer (MS)IonizerIonizer In the GC-MS discussed in this introduction, the charged particles (ions) In the GC-MS discussed in this introduction, the charged particles (ions)

required for mass analysis are formed by Electron Impact (EI) Ionization. required for mass analysis are formed by Electron Impact (EI) Ionization. The gas molecules exiting the GC are bombarded by a high-energy electron The gas molecules exiting the GC are bombarded by a high-energy electron beam (70 eV). An electron which strikes a molecule may impart enough beam (70 eV). An electron which strikes a molecule may impart enough energy to remove another electron from that molecule. Methanol, for energy to remove another electron from that molecule. Methanol, for example, would undergo the following reaction in the ionizing region: example, would undergo the following reaction in the ionizing region: CH3OH + 1 electron CH3OH+CH3OH + 1 electron CH3OH+..+ 2 electrons + 2 electrons (note: the symbols (note: the symbols +.+. indicate that a radical cation was formed) indicate that a radical cation was formed)

EI Ionization usually produces singly charged ions containing one unpaired EI Ionization usually produces singly charged ions containing one unpaired electron. A charged molecule which remains intact is called the molecular electron. A charged molecule which remains intact is called the molecular ion. Energy imparted by the electron impact and, more importantly, ion. Energy imparted by the electron impact and, more importantly, instability in a molecular ion can cause that ion to break into smaller pieces instability in a molecular ion can cause that ion to break into smaller pieces (fragments). The methanol ion may fragment in various ways, with one (fragments). The methanol ion may fragment in various ways, with one fragment carrying the charge and one fragment remaining uncharged. For fragment carrying the charge and one fragment remaining uncharged. For example: example: CH3OH+CH3OH+..(molecular ion) CH2OH+(fragment ion) + H(molecular ion) CH2OH+(fragment ion) + H.. (or) CH3OH+(or) CH3OH+..(molecular ion) CH3+(fragment ion) + (molecular ion) CH3+(fragment ion) + ..OHOH


Ion AnalyzerIon Analyzer Molecular ions and fragment ions are accelerated by manipulation Molecular ions and fragment ions are accelerated by manipulation

of the charged particles through the mass spectrometer. of the charged particles through the mass spectrometer. Uncharged molecules and fragments are pumped away. The Uncharged molecules and fragments are pumped away. The quadrupole mass analyzer in this example uses positive (+) and quadrupole mass analyzer in this example uses positive (+) and negative (-) voltages to control the path of the ions. Ions travel negative (-) voltages to control the path of the ions. Ions travel down the path based on their mass to charge ratio (m/z). EI down the path based on their mass to charge ratio (m/z). EI ionization produces singly charged particles, so the charge (z) is ionization produces singly charged particles, so the charge (z) is one. Therefore an ion's path will depend on its mass. If the (+) and one. Therefore an ion's path will depend on its mass. If the (+) and (-) rods shown in the mass spectrometer schematic were ?fixed' at a (-) rods shown in the mass spectrometer schematic were ?fixed' at a particular rf/dc voltage ratio, then one particular m/z would travel the particular rf/dc voltage ratio, then one particular m/z would travel the successful path shown by the solid line to the detector. However, successful path shown by the solid line to the detector. However, voltages are not fixed, but are scanned so that ever increasing voltages are not fixed, but are scanned so that ever increasing masses can find a successful path through the rods to the detector. masses can find a successful path through the rods to the detector.


DetectorDetector There are many types of detectors, but most work by There are many types of detectors, but most work by

producing an electronic signal when struck by an ion. producing an electronic signal when struck by an ion. Timing mechanisms which integrate those signals with Timing mechanisms which integrate those signals with the scanning voltages allow the instrument to report the scanning voltages allow the instrument to report which m/z strikes the detector. The mass analyzer sorts which m/z strikes the detector. The mass analyzer sorts the ions according to m/z and the detector records the the ions according to m/z and the detector records the abundance of each m/z. Regular calibration of the m/z abundance of each m/z. Regular calibration of the m/z scale is necessary to maintain accuracy in the scale is necessary to maintain accuracy in the instrument. Calibration is performed by introducing a instrument. Calibration is performed by introducing a well known compound into the instrument and "tweaking" well known compound into the instrument and "tweaking" the circuits so that the compound's molecular ion and the circuits so that the compound's molecular ion and fragment ions are reported accurately. fragment ions are reported accurately.

Protein Structure PredictionProtein Structure Prediction

is the prediction of the three-dimensional is the prediction of the three-dimensional structure of a protein from its amino acid structure of a protein from its amino acid sequence—that is, the prediction of a protein's sequence—that is, the prediction of a protein's tertiary structure from its primary structure. It is tertiary structure from its primary structure. It is one of the most important goals pursued by one of the most important goals pursued by bioinformatics and theoretical chemistry. Protein bioinformatics and theoretical chemistry. Protein structure prediction is of high importance in structure prediction is of high importance in medicine (for example, in drug design) and medicine (for example, in drug design) and biotechnology (for example, in the design of biotechnology (for example, in the design of novel enzymes). novel enzymes).

Protein SequencingProtein Sequencing Proteins are found in every cell and are essential to every biological Proteins are found in every cell and are essential to every biological

process, protein structure is very complex: determining a protein's process, protein structure is very complex: determining a protein's structure involves first structure involves first protein sequencingprotein sequencing - determining the amino - determining the amino acid sequences of its constituent peptides; and also determining acid sequences of its constituent peptides; and also determining what conformation it adopts and whether it is complexed with any what conformation it adopts and whether it is complexed with any non-peptide molecules. Discovering the structures and functions of non-peptide molecules. Discovering the structures and functions of proteins in living organisms is an important tool for understanding proteins in living organisms is an important tool for understanding cellular processes, and allows drugs that target specific metabolic cellular processes, and allows drugs that target specific metabolic pathways to be invented more easily.pathways to be invented more easily.

The two major direct methods of protein sequencing are The two major direct methods of protein sequencing are mass mass spectrometryspectrometry and the and the Edman degradation reactionEdman degradation reaction. It is also . It is also possible to generate an amino acid sequence from the DNA or possible to generate an amino acid sequence from the DNA or mRNA sequence encoding the protein, if this is known. However, mRNA sequence encoding the protein, if this is known. However, there are a number of other reactions which can be used to gain there are a number of other reactions which can be used to gain more limited information about protein sequences and can be used more limited information about protein sequences and can be used as preliminaries to the aforementioned methods of sequencing or to as preliminaries to the aforementioned methods of sequencing or to overcome specific inadequacies within them.overcome specific inadequacies within them.

Protein SequencingProtein Sequencing

Edman degradationEdman degradation: is a method of : is a method of sequencing amino acids in a peptide. In sequencing amino acids in a peptide. In this method, the amino-terminal residue is this method, the amino-terminal residue is labeled and cleaved from the peptide labeled and cleaved from the peptide without disrupting the peptide bonds without disrupting the peptide bonds between other amino acid residues.between other amino acid residues.

Protein SequencingProtein Sequencing PhenylisothiocyanatePhenylisothiocyanate is reacted with an uncharged terminal amino is reacted with an uncharged terminal amino

group, under mildly alkaline conditions, to form a cyclical group, under mildly alkaline conditions, to form a cyclical phenylthiocarbamoylphenylthiocarbamoyl derivative. Then, under acidic conditions, this derivative. Then, under acidic conditions, this derivative of the terminal amino acid is cleaved as a thiazolinone derivative of the terminal amino acid is cleaved as a thiazolinone derivative. The thiazolinone amino acid is then selectively extracted derivative. The thiazolinone amino acid is then selectively extracted into an organic solvent and treated with acid to form the more stable into an organic solvent and treated with acid to form the more stable phenylthiohydantoin (PTH)- amino acid derivative that can be phenylthiohydantoin (PTH)- amino acid derivative that can be identified by using chromatography or electrophoresis. identified by using chromatography or electrophoresis.

This procedure can then be repeated again to identify the next amino This procedure can then be repeated again to identify the next amino acid. A major drawback to this technique is that the peptides being acid. A major drawback to this technique is that the peptides being sequenced in this manner cannot have more than 50 to 60 residues sequenced in this manner cannot have more than 50 to 60 residues (and in practice, under 30). The peptide length is limited due to the (and in practice, under 30). The peptide length is limited due to the cyclical derivitization not always going to completion. The cyclical derivitization not always going to completion. The derivitization problem can be resolved by cleaving large peptides into derivitization problem can be resolved by cleaving large peptides into smaller peptides before proceeding with the reaction. It is able to smaller peptides before proceeding with the reaction. It is able to accurately sequence up to 30 amino acids with modern machines accurately sequence up to 30 amino acids with modern machines capable of over 99% efficiency per amino acid. An advantage of the capable of over 99% efficiency per amino acid. An advantage of the Edman degradation is that it only uses 10 - 100 picomoles of peptide Edman degradation is that it only uses 10 - 100 picomoles of peptide for the sequencing process. Edman degradation reaction is for the sequencing process. Edman degradation reaction is automated to speed up the process. automated to speed up the process.

Protein SequencingProtein Sequencing

Protein Structural AlignmentsProtein Structural Alignments Structural alignmentStructural alignment is a form of sequence alignment that is based is a form of sequence alignment that is based

on comparison of shape. These alignments attempt to establish on comparison of shape. These alignments attempt to establish equivalences between two or more polymer structures based on equivalences between two or more polymer structures based on their shape and three-dimensional conformation. This process is their shape and three-dimensional conformation. This process is usually applied to protein tertiary structures but can also be used for usually applied to protein tertiary structures but can also be used for large RNA molecules. In contrast to simple structural superposition, large RNA molecules. In contrast to simple structural superposition, where at least some equivalent residues of the two structures are where at least some equivalent residues of the two structures are known, structural alignment requires no known, structural alignment requires no a prioria priori knowledge of knowledge of equivalent positions. Structural alignment is a valuable tool for the equivalent positions. Structural alignment is a valuable tool for the comparison of proteins with low sequence similarity, where comparison of proteins with low sequence similarity, where evolutionary relationships between proteins cannot be easily evolutionary relationships between proteins cannot be easily detected by standard sequence alignment techniques. Structural detected by standard sequence alignment techniques. Structural alignment can therefore be used to imply evolutionary relationships alignment can therefore be used to imply evolutionary relationships between proteins that share very little common sequence. However, between proteins that share very little common sequence. However, caution should be used in using the results as evidence for shared caution should be used in using the results as evidence for shared evolutionary ancestry because of the possible confounding effects evolutionary ancestry because of the possible confounding effects of convergent evolution by which multiple unrelated amino acid of convergent evolution by which multiple unrelated amino acid sequences converge on a common tertiary structure.sequences converge on a common tertiary structure.

Protein Structural AlignmentsProtein Structural Alignments Structural alignments can compare two sequences or Structural alignments can compare two sequences or multiple sequencesmultiple sequences. .

Because these alignments rely on information about all the query Because these alignments rely on information about all the query sequences' three-dimensional conformations, the method can only be used sequences' three-dimensional conformations, the method can only be used on sequences where these structures are known. These are usually found on sequences where these structures are known. These are usually found by by X-ray crystallographyX-ray crystallography or or NMR spectroscopyNMR spectroscopy. It is possible to perform a . It is possible to perform a structural alignment on structures produced by structural alignment on structures produced by structure predictionstructure prediction methods. methods. Indeed, evaluating such predictions often requires a structural alignment Indeed, evaluating such predictions often requires a structural alignment between the model and the true known structure to assess the model's between the model and the true known structure to assess the model's quality. Structural alignments are especially useful in analyzing data from quality. Structural alignments are especially useful in analyzing data from structural structural genomicsgenomics and and proteomicsproteomics efforts, and they can be used as efforts, and they can be used as comparison points to evaluate alignments produced by purely sequence-comparison points to evaluate alignments produced by purely sequence-based based bioinformaticsbioinformatics methods. methods.

The outputs of a structural alignment are a superposition of the atomic The outputs of a structural alignment are a superposition of the atomic coordinate setscoordinate sets and a minimal and a minimal root mean squareroot mean square distance ( distance (RMSDRMSD) between ) between the structures. The RMSD of two aligned structures indicates their the structures. The RMSD of two aligned structures indicates their divergence from one another. Structural alignment can be complicated by divergence from one another. Structural alignment can be complicated by the existence of multiple the existence of multiple protein domainsprotein domains within one or more of the input within one or more of the input structures, because changes in relative orientation of the domains between structures, because changes in relative orientation of the domains between two structures to be aligned can artificially inflate the RMSD. two structures to be aligned can artificially inflate the RMSD.

Protein Structural AlignmentsProtein Structural Alignments

METHODS:METHODS: GANGSTAGANGSTA ( (GGenetic enetic AAlgorithm for lgorithm for NNon-sequential, on-sequential,

GGapped protein apped protein STSTructure ructure AAlignment) is a method for lignment) is a method for non-sequential protein structure alignment using a two-non-sequential protein structure alignment using a two-level hierarchical approach. On the first level, pairwise level hierarchical approach. On the first level, pairwise contacts and relative orientations between SSEs are contacts and relative orientations between SSEs are maximized using a genetic algorithm (GA) and protein maximized using a genetic algorithm (GA) and protein graph representation. On the second level, pairwise graph representation. On the second level, pairwise residue contact maps resulting from the best SSE residue contact maps resulting from the best SSE alignments are optimized. GANGSTA can be used online alignments are optimized. GANGSTA can be used online at at http://agknapp.chemie.fu-berlin.de/gangstahttp://agknapp.chemie.fu-berlin.de/gangsta

Protein Structural AlignmentsProtein Structural Alignments

METHODS:METHODS: MAMMOTH (MAMMOTH (MAtching Molecular Models Obtained MAtching Molecular Models Obtained

from Theory) from Theory) -based structure alignment methods -based structure alignment methods decomposes the protein structure into short peptides decomposes the protein structure into short peptides (heptapeptides) which are compared with the (heptapeptides) which are compared with the heptapeptides of another protein. Similarity score heptapeptides of another protein. Similarity score between two heptapeptides is calculated using a unit-between two heptapeptides is calculated using a unit-vector RMS (URMS) method. These scores are stored in vector RMS (URMS) method. These scores are stored in a similarity matrix, and with a hybrid (local-global) a similarity matrix, and with a hybrid (local-global) dynamic programming the optimal residue alignment is dynamic programming the optimal residue alignment is calculated. Protein similarity scores calculated with calculated. Protein similarity scores calculated with MAMMOTH is derived from the likelihood of obtaining a MAMMOTH is derived from the likelihood of obtaining a given structural alignment by chance given structural alignment by chance

Protein Structural AlignmentsProtein Structural Alignments RAPIDORAPIDO (R (Rapid apid AAlignment of lignment of PProteins roteins IIn terms of n terms of DoDomains) is a web server mains) is a web server

for the 3D alignment of crystal structures of different protein molecules, in the for the 3D alignment of crystal structures of different protein molecules, in the presence of conformational changes. RAPIDO identifies fragments that are presence of conformational changes. RAPIDO identifies fragments that are structurally similar in the two proteins using an approach based on difference structurally similar in the two proteins using an approach based on difference distance matrices. The Matching Fragment Pairs (MFPs) are then distance matrices. The Matching Fragment Pairs (MFPs) are then represented as nodes in a graph which are chained together to form an represented as nodes in a graph which are chained together to form an alignment by means of an algorithm for the identification of the longest path alignment by means of an algorithm for the identification of the longest path on a DAG (Directed Acyclic Graph). The final step of refinement is performed on a DAG (Directed Acyclic Graph). The final step of refinement is performed to improve the quality of the alignment. After aligning the two structures the to improve the quality of the alignment. After aligning the two structures the server applies a genetic algorithm for the identification of conformationally server applies a genetic algorithm for the identification of conformationally invariant regions . These regions correspond to groups of atoms whose invariant regions . These regions correspond to groups of atoms whose interatomic distances are constant (within a defined tolerance). In doing so interatomic distances are constant (within a defined tolerance). In doing so RAPIDO takes into account the variation in the reliability of atomic RAPIDO takes into account the variation in the reliability of atomic coordinates by employing weighting-functions based on the refined B-values. coordinates by employing weighting-functions based on the refined B-values. The regions identified as conformationally invariant by RAPIDO represent The regions identified as conformationally invariant by RAPIDO represent reliable sets of atoms for the superposition of the two structures that can be reliable sets of atoms for the superposition of the two structures that can be used for a detailed analysis of changes in the conformation. In addition to the used for a detailed analysis of changes in the conformation. In addition to the functionalities provided by existing tools, RAPIDO can identify structurally functionalities provided by existing tools, RAPIDO can identify structurally equivalent regions even when these consist of fragments that are distant in equivalent regions even when these consist of fragments that are distant in terms of sequence and separated by other movable domains.terms of sequence and separated by other movable domains.

Protein Structural AlignmentsProtein Structural Alignments SABERTOOTHSABERTOOTH uses structural profiles to perform structural uses structural profiles to perform structural

alignments. The underlying structural profiles expresses the global alignments. The underlying structural profiles expresses the global connectivity of each residue. Despite the very condensed vectorial connectivity of each residue. Despite the very condensed vectorial representation, the tool recognizes structural similarities with representation, the tool recognizes structural similarities with accuracy comparable to established alignment tools based on accuracy comparable to established alignment tools based on coordinates and performs comparably in quality. Furthermore, the coordinates and performs comparably in quality. Furthermore, the algorithm has favourable scaling of computation time with chain algorithm has favourable scaling of computation time with chain length. Since the algorithm is independent of the details of the length. Since the algorithm is independent of the details of the structural representation, the framework can be generalized to structural representation, the framework can be generalized to sequence-to-sequence and sequence-to-structure comparison sequence-to-sequence and sequence-to-structure comparison within the same setup, and it is therefore more general than other within the same setup, and it is therefore more general than other tools. SABERTOOTH can be used online at http://www.fkp.tu-tools. SABERTOOTH can be used online at http://www.fkp.tu-darmstadt.de/sabertooth/darmstadt.de/sabertooth/

Peptide Mass FingerprintingPeptide Mass Fingerprinting Peptide mass fingerprintingPeptide mass fingerprinting (PMF) (also known as (PMF) (also known as

protein fingerprintingprotein fingerprinting) is an analytical technique for protein ) is an analytical technique for protein identification that was developed in 1993 by several groups identification that was developed in 1993 by several groups independently. In this method, the unknown protein of independently. In this method, the unknown protein of interest is first cleaved into smaller interest is first cleaved into smaller peptidespeptides, whose , whose absolute masses can be accurately measured with a absolute masses can be accurately measured with a mass spectrometermass spectrometer such as such as MALDI-TOFMALDI-TOF or or ESI-TOFESI-TOF. . These masses are then These masses are then in in silicosilico compared to either a compared to either a database containing known protein sequences or even the database containing known protein sequences or even the genome. This is achieved by using computer programs that genome. This is achieved by using computer programs that translate the known genome of the organism into proteins, translate the known genome of the organism into proteins, then theoretically cut the proteins into peptides, and then theoretically cut the proteins into peptides, and calculate the absolute masses of the peptides from each calculate the absolute masses of the peptides from each protein. They then compare the masses of the peptides of protein. They then compare the masses of the peptides of the unknown protein to the theoretical peptide masses of the unknown protein to the theoretical peptide masses of each protein encoded in the genome. The results are each protein encoded in the genome. The results are statistically analyzed to find the best match. statistically analyzed to find the best match.

Peptide Mass FingerprintingPeptide Mass Fingerprinting The advantage of this method is that only the masses of the The advantage of this method is that only the masses of the

peptides have to be known and as such, peptides have to be known and as such, de novode novo peptide sequencing peptide sequencing is not necessary which can be time consuming. is not necessary which can be time consuming. A disadvantage is that the protein sequence has to be present in the A disadvantage is that the protein sequence has to be present in the database of interest. Additionally most PMF algorithms assume that database of interest. Additionally most PMF algorithms assume that the peptides come from a single protein. The presence of a mixture the peptides come from a single protein. The presence of a mixture can significantly complicate the analysis and potentially compromise can significantly complicate the analysis and potentially compromise the results. Typical for the PMF based protein identification is the the results. Typical for the PMF based protein identification is the requirement for an isolated protein. Mixtures exceeding a number of requirement for an isolated protein. Mixtures exceeding a number of 2-3 proteins typically require the additional use of 2-3 proteins typically require the additional use of MS/MSMS/MS based based protein identification to achieve sufficient specificity of identification. protein identification to achieve sufficient specificity of identification. Therefore, the typical PMF samples are isolated proteins from Therefore, the typical PMF samples are isolated proteins from Two-dimensional gel electrophoresisTwo-dimensional gel electrophoresis (2D gels) or isolated (2D gels) or isolated SDS-PAGESDS-PAGE bands. Additional analyses by bands. Additional analyses by MS/MSMS/MS can either be can either be direct, e.g., MALDI-TOF/TOF analysis or downstream nanoLC-ESI-direct, e.g., MALDI-TOF/TOF analysis or downstream nanoLC-ESI-MS/MS analysis of gel spot eluates. MS/MS analysis of gel spot eluates.

Ligand Binding AssayLigand Binding Assay a a ligand binding assayligand binding assay is technique to identify is technique to identify

the presence of a molecule and quantify it.the presence of a molecule and quantify it. Historically, it was used to test for estrogen Historically, it was used to test for estrogen

receptor and progesterone receptor in breast receptor and progesterone receptor in breast tumorstumors

Characterization through ligand interaction may Characterization through ligand interaction may be performed by electroblotting or by affinity be performed by electroblotting or by affinity electrophoresis in agarose or by capillary electrophoresis in agarose or by capillary electrophoresis as for estimation of binding electrophoresis as for estimation of binding constants and determination of structural constants and determination of structural features like glycan content through lectin features like glycan content through lectin binding. binding.

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Protein Detection Methods and Application

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