Proteins (Isolation Techniques).pdf
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Transcript of Proteins (Isolation Techniques).pdf
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CELL AND MOLECULAR BIOLOGY
Prepared by: Jassy Mary S. Lazarte & Jeremy G. Vicencio
Department of Biology, University of the Philippines ManilaBio150 Lab
PROTEINS
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What are Proteins?
Protein from the Greek proteios (primary, first, and
foremost)
Proteins are the work horses of biological systems.
They play key roles in constructing and maintaining livingcells
Our genes code for proteins
Proteins are polymers of amino acids
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Proteins are
Polypeptides + (cofactors, coenzymes, prosthetic
groups, other modifications)
Polypeptides are covalently linked -amino acids
Cofactors are non-amino acid components e.g. metal ionslike Zn2+ in carboxypeptidase
Coenzymes are organic cofactors e.g. nucleotides in lactate
dehydrogenase
Prosthetic groups are covalently attached cofactors e.g.heme in myoglobin
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Proteins and their roles
Enzymes (biological catalysts)
Hormones
Storage proteins
Transport proteins
Structural proteins
Protective proteins
Contractile proteins
Toxic proteins
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Proteins and their structure
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How to isolate proteins?
1. Lyse the cell
2. Solubilize the proteins
use detergents in the protein extraction buffer
a. Nonionic detergents (milder)
ex. Triton X-100: break lipid-lipid interaction and
lipid-protein interaction
b. Anionic detergents (more denaturing)ex. SDS: protein-protein interaction,Sodium Deoxycholate: protein-protein interaction
*Upon lysis of the cell, proteases are released into the
lysate
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Protein Isolation
What are proteases?
aka proteinases, peptidases or proteolytic enzymes
are enzymes that break peptide bonds between amino
acids of proteins Where do proteases come from?
Animal cells: Lysosomes, contain a large variety of hydrolytic
enzymes that degrade proteins and other substances
Plant cells: Vacuole, many hydrolytic enzymes found invacuole resemble those present in Lysosomes of animal cells
other organelles also have proteases
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Protein Isolation
How to prevent protein degradation by
proteases
the protein isolation is carried out at low
temperature to minimize the activities of theseprotease
to further optimize the results, use the proteases
inhibitors
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Protein Isolation
3. Addition of Protease Inhibitors
EDTA (or EGTA): chelating the Ca2+
PMSF: a general serine protease inhibitor.
*It is the most common inhibitor used in protein purification.
*Soluble in isopropanol.
4. Quantify Protein
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Protein Quantitation
A. Colorimetric methods:
1. Biuret
2. Lowry
3. BradfordB. UV absorption method
*The amino acids tryptophan, tyrosine and phenylalanine absorb
light in the UV wavelength
Since the absorption is proportional to concentration, this is a
useful way to quantitate protein concentration (for proteins
containing Trp)
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Protein Quantitation
Disadvantages of UV absorption method
If some proteins do not contain these amino acids, it will
not absorb UV light
Nucleic acids (DNA, RNA) contaminant will also absorbUV light
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Protein Quantitation
A260/A280 has high
sensitivity for nucleic acid
contamination in protein:
A260/A280 lacks sensitivity
for protein contamination
in nucleic acids:
% protein % NA A260/A280
100 0 0.57
95 5 1.0690 10 1.32
70 30 1.73
% NA % protein A260/A280
100 0 2.00
95 5 1.9990 10 1.98
70 30 1.94
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Protein Quantitation
Colorimetric methods
protein samples can be modified with appropriate
reagents to produce a color reaction and measure
protein concentration using a spectrophotometer.
Advantages of Colorimetric methods1. Cheap cuvette! (cheap glass or plastic versus
quartz)2. Not contaminating absorbance from nucleic acids!
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BRADFORD ASSAY
Bradford Method
A dye known as CBBG, Coomassie
Brilliant Blue G-250 was developed
by the textile industry. It was noticed to stain skin as well as
the textiles.
This dye (which normally absorbs at465nm) binds to proteins and to
absorb strongly at 595nm.
The assay is sensitive, but somewhat
non-linear
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Dye-Binding ( Bradford ) Assay
Bradford, MM. A rapid and sensitive for the quantitation of microgram
quantitites of protein utilizing the principle of protein-dye binding.AnalyticalBiochemistry 72:248-254. 1976.
Stoscheck, CM. Quantitation of Protein. Methods in Enzymology 182:50-69(1990).
CBBG primarily responds toarginine residues (eight times as
much as the other listed residues)
If you have an arginine rich protein,
You may need to find a standard
that is arginine rich as well.
CBBG binds to these residues in theanionic form
Absorbance maximum at 595 nm(blue)
The free dye in solution is in thecationic form,
Absorbance maximum at 470 nm
(red).
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Dye-Binding ( Bradford ) Assay
Advantages Fast and inexpensiveHighly specific for protein
Very sensitive [1-20 g (micro assay) 20-200 g(macro assay)]
Compatible with a wide range of substances Extinction co-efficient for the dye-protein complex is
stable over 10 orders of magnitude (assessed inalbumin)Dye reagent is complex is stable for approximately
one hour
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Dye-Binding ( Bradford ) Assay
DisadvantagesNon-linear standard curve over wide rangesResponse to different proteins can vary widely,
choice of standard is very important
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Dye-Binding ( Bradford ) Assay
Absorption spectra of anionic and cationic forms of thedye overlap.
So the standard curve is non-linear although all kitproviders of the Bradford assay insist that the assayperforms linearly.
The assay performs linearly over short concentrationstretches.
If your sample is more than 20 micrograms, a secondorder curve will fit much better than a linear curve.
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Points to remember:
Why measure absorbance at 595nm?
When the dye reacts with the protein(protein-dye complex), a
blue-colored solution will be formed which optimally absorbs
light at such wavelength Why should incubation not exceed 1 hour?
The dye interacts with the protein resulting to formation of
precipitates which might give an inaccurate absorbance
reading
What is the relationship of the absorbance reading
with the protein concentration? Directly proportional
What is the identity of the standards? bovine -globulins
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Hydrophobic Interaction Chromatography
Hydrophobic interaction - association of nonpolar
molecules that minimizes exposure to the polar
surroundings
Chromatography - a technique used in which amixture of dissolved components is fractionated as it
moves through a certain type of porous matrix
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HIC
Alternatives
Gel filtration chromatography
Ion exchange chromatography
Reverse phase chromatography
Why HIC?
Different basis of separation
Weaker interactions Less structural damage
Maintain high activity
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Separation principles
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HIC: Purpose
Downstream purification
Separation of biomolecules
Exploits differences in hydrophobicity.
Number of hydrophobic amino acids.
Distribution of these amino acids.
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HIC: Principle
Separation of substances is based on their
varying strength of interaction with
hydrophobic groups attached to an uncharged
gel matrix
Hydrophobic groups on proteins are sufficiently
exposed to bind to the hydrophobic groups on
the matrix. How is this achieved?
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HIC: Principle
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HIC: Principle
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HIC: Applications
HIC has found many applications in the purification of
a wide variety of proteins, including
Monoclonal antibodies Vaccines
Truncated forms of r-proteins
Interferons EGF (epidermal growth factor)
Human growth hormone
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HIC: Advantages
Large volume of sample can be loaded
Samples with high ionic strength can be used
Well suited to use before gel filtration, ion-
exchange and affinity chromatography
Sample eluted with low salt
Purification steps that generate large sample
volume can be coupled with this method
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HIC: Advantages
Good for samples after ammonium sulfate
fractionation.
Sample can be used in ion exchange
chromatography step
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Factors affecting HIC
Type and concentration of ligand
Type of base matrix
Type and concentration of salt
pH
Temperature
Additives
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Buffers
Equilibration buffer, 2 M (NH4)2SO4- used to prime the
column for the binding of proteins
Binding buffer, 4 M (NH4)2SO4- hydrophobic region is
more exposed causing it to interact and bind with thehydrophobic regions of the matrix
Wash buffer, 1.3 M (NH4)2SO4-use - wash the weakly
associated proteins from the column, while the strongly
hydrophobic proteins are bound to the matrixElution buffer, 10 mM Tris/EDTA, -wash the strongly
hydrophobic proteins from the column
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Thus in the fractions obtained
1st fraction: hydrophilic proteins
2nd fraction: the rest of hydrophilic plus some
hydrophobic proteins 3rd fraction: strongly hydrophobic proteins
the last fraction that has the highest absorbance
reading contains the most hydrophobic proteins
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Enzyme-linked Immunosorbent Assay
ELISA
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What is an ELISA?
Enzyme-linked immunosorbent assay
Name suggests three components
Antibody
Allows for specific detection of analyte of interest Solid phase (sorbent) Allows one to wash away all the material that is not
specifically captured
Enzymatic amplification Allows you to turn a little capture into a visible color change
that can be quantified using an absorbance plate reader
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What is it used for?
Measure antibody levels (allergies, vaccines)
Detect viruses (hepatitis, HIV, venereal diseases)
Detect hormonal changes (pregnancy)
Detect circulatory inflammatory markers (cytokines)
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Advantages
Sensitivity
Quantitative
Reproducible
Kit format
Relative sensitivities of tests (approx)
Usual operating range
[Ab] or [Ag]
precipitation
immunoelectrophoresis
double/radial diffusion
10 g/ml - 1 mg/ml
immunofluorescence 0.1 - 10 g/ml
ELISA (colour) 0.1 - 10 ng/ml
(chemiluminescence) 0.01 - 10 ng/ml
radioimmunoassay 0.01 - 10 ng/ml
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Enzymes with Chromogenic Substrates
High molar extinction coefficient (i.e., strong color
change)
Strong binding between enzyme and substrate (low
KM) Linear relationship between color intensity and
[enzyme]
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Antibodies
Specificity Diversityhypervariable region (2020 ~ 1026
combinations; humans make ~ 108)
Affinityrange 105 < K < 109 M-1
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Capture and Detection Antibodies
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ELISA
Enzyme Linked Immunosorbent Assay (ELISA)
Term Was Coined By Engvall and Pearlmann in 1971
Different Types
Sandwich
Indirect
Competitive
Similar To RIA(radio-immuno assay), Except No Radiolabel Can Be Used To Detect Both Antibody and Antigen
Very Sensitive, pg/mL
Relies on Monoclonal Abs
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ELISA-Types
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ELISA
Enzyme-linked immunosorbent assay
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ELISA: In the experiment..
REAGENTS:
Ag: chicken -globulin
1Ab: rabbit anti-chicken polyclonal Ab
2Ab: goat Ab conjugated to horseradish
peroxidase(HRP)
-substrate: 3,3,5,5-tetrametylbenzidine(TMB)
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ELISA: In the experiment
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ELISA-In the experiment..
SPECIFICS:
1. Wash buffer: to discard unbounded Ag
2. Incubation: to allow the Abs react with the Ag
3. HRP enzyme substrate- gives color
*microtiter plates: polystyrene
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ELISA-In the experiment..
RESULTS
amount of colored product is proportional to the
amount of enzyme-linked antibody that binds, which is
directly related to the amount of antibody that waspresent to bind antigen or antigen that was present to
bind antibody
If known amounts of antigen or antibody are added,
a standard curve can be constructed which will allow
the amount of unknown antigen or antibody to be
determined