Gel electrophoresis

Gel electrophoresisGel electrophoresis separates molecules on the basis of their charge and size. The charged macromolecules migrate across a span of gel because they are placed in an electrical field. The gel acts as a sieve to retard the passage of molecules according to their size and shape.Gel electrophoresis is a technique used for the separation of deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or protein molecules using an electric current applied to a gel matrix. "Electrophoresis" refers to the electromotive force (EMF) that is used to move the molecules through the gel matrix. By placing the molecules in wells in the gel and applying an electric current, the molecules will move through the matrix at different rates, determined largely by their mass when the charge to mass ratio (Z) of all species is uniform, toward the anode if negatively charged or toward the cathode if positively charged.

• Om's Law and Electrophoresis • Understanding how a gel apparatus is

connected to the power supply requires a basic understanding om's law: voltage = current x resistance, or V = I X R. A gel can be viewed as a resistor and the power supply as the voltage and current source. Most power supplies deliver constant current or constant source. Some will also deliver constant power: power = voltage x current;

• Most modem commercial euquipment is color-coded so that the red or positive terminal of the powerterminal simply be connected to the red lead of the gel apparatus, which goes to the lower buffer chamber. The black lead is connected to the black: or negative terminal and goes to the upper buffer chamber. This configuration is designed to work: with vertical slab gel electrophoreses in which nega tively charged proteins or nucleic acids move to the positive electrode in the lower buffer chamber (anionic system).

• When a single gel is attached to a power supply in an anionic system, the negative charges flow from negative cathode (black) terminal into the upper buffer chamber, through the gel, and into the lower buffer chamber, which is connected to the positive anode (red) terminal to complete the circuit. ·PAGE is an anionic system because of the negatively charged SDS. Occasionally, proteins are separeted in cationic systems. In these gels, the proteins are positively charged because of the very pH of the gel buffers or the presence of a cationic detergent (e.g., cetyltrimethylammonium bro ,CTAB). Proteins move toward the negetive electrode (cathode), and the polarity is reversed pared to SDS-PAGE (the red lead from the lower buffer chamber is attached to the black outlet of power supply).

SeparationSeparation

The term "The term "gel" in this instance refers to the matrix used to " in this instance refers to the matrix used to contain, then separate the target molecules. In most cases, the contain, then separate the target molecules. In most cases, the gel is a gel is a crosslinked polymer whose composition and porosity is whose composition and porosity is chosen based on the specific weight and composition of the chosen based on the specific weight and composition of the target to be analyzed. When separating target to be analyzed. When separating proteins or small or small nucleic acids ( (DNA, , RNA, or , or oligonucleotides) the gel is usually ) the gel is usually composed of different concentrations of composed of different concentrations of acrylamide and a and a cross-linker, producing different sized mesh networks of , producing different sized mesh networks of polyacrylamide. When separating larger nucleic acids (greater . When separating larger nucleic acids (greater than a few hundred than a few hundred bases), the preferred matrix is purified ), the preferred matrix is purified agarose. In both cases, the gel forms a solid, yet porous matrix. . In both cases, the gel forms a solid, yet porous matrix. Acrylamide, in contrast to , in contrast to polyacrylamide, is a , is a neurotoxin and and must be handled using appropriate safety precautions to avoid must be handled using appropriate safety precautions to avoid poisoning. poisoning. Agarose is composed of long unbranched chains of is composed of long unbranched chains of uncharged carbohydrate without cross links resulting in a gel uncharged carbohydrate without cross links resulting in a gel with large pores allowing for the separation of macromolecules with large pores allowing for the separation of macromolecules and and macromolecular complexes

After the electrophoresis is complete, the molecules in the gel can be stained to make them visible. Ethidium bromide, silver, or coomassie blue dye may be used for this process. Other methods may also be used to visualize the separation of the mixture's components on the gel. If the analyte molecules fluoresce under ultraviolet light, a photograph can be taken of the gel under ultraviolet lighting conditions. If the molecules to be separated contain radioactivity added for visibility, an autoradiogram can be recorded of the gel. If several mixtures have initially been injected next to each other, they will run parallel in individual lanes. Depending on the number of different molecules, each lane shows separation of the components from the original mixture as one or more distinct bands, one band per component. Incomplete separation of the components can lead to overlapping bands, or to indistinguishable smears representing multiple unresolved components.

Bands in different lanes that end up at the same distance from the top contain molecules that passed through the gel with the same speed, which usually means they are approximately the same size. There are molecular weight size markers available that contain a mixture of molecules of known sizes. If such a marker was run on one lane in the gel parallel to the unknown samples, the bands observed can be compared to those of the unknown in order to determine their size. The distance a band travels is approximately inversely proportional to the logarithm of the size of the molecule.

Nucleic acidsIn the case of nucleic acids, the direction of migration, from negative to positive electrodes, is due to the naturally-occurring negative charge carried by their sugar-phosphate backbone.Double-stranded DNA fragments naturally behave as long rods, so their migration through the gel is relative to their size . Single-stranded DNA or RNA tend to fold up into molecules with complex shapes and migrate through the gel in a complicated manner based on their tertiary structure. Therefore, agents that disrupt the hydrogen bonds, such as sodium hydroxide or formamide, are used to denature the nucleic acids and cause them to behave as long rods again.Gel electrophoresis of large DNA or RNA is usually done by agarose gel electrophoresis.

ProteinsProteins, unlike nucleic acids, can have varying charges

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

Proteins are usually analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), by native gel electrophoresis, by quantitative preparative native continuous polyacrylamide gel electrophoresis (QPNC-PAGE), or by 2-D electrophoresis.Characterization through ligand interaction may be performed by electroblotting or by affinity electrophoresis in agarose or by capillary electrophoresis as for estimation of binding constants and determination of structural features like glycan content through lectin binding.

Native Gel Electrophoresis is a technique used mainly in protein electrophoresis where the proteins are not denatured and therefore separated based on their charge-to-mass ratio. Polyacrylamide gel electrophoresis (PAGE) is used for separating proteins ranging in size from 5 to 200 kiloDalton due to the uniform pore size provided by the polyacrylamide gel. Pore size is controlled by controlling the concentrations of acrylamide and bis-acrylamide powder used in creating a gel. Care must be used when creating this type of gel, as acrylamide is a potent neurotoxin in its liquid and powdered form. The other type of gel used is agarose gel. Agarose gels can also be used to separate native protein. They do not have a uniform pore size, but are optimal for electrophoresis of proteins that are larger than 200 kDalton.

Unlike SDS-PAGE type electrophoreses, Native gel electrophoresis does not use a charged denaturing agent. The molecules being separated (usually proteins) therefore differ in Molecular mass and intrinsic charge and experience different electrophoretic forces dependent on the ratio of the two. Since the proteins remain in the native state they may be visualised not only by general protein staining reagents but also by specific enzyme-linked staining . SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique widely used in biochemistry, forensics, genetics and molecular biology to separate proteins according to their electrophoretic mobility. The SDS gel electrophoresis of samples having identical charge per unit mass due to binding of SDS results in fractionation by size and is probably the world's most widely used biochemical method.

ProcedureThe solution of proteins to be analyzed is first mixed with SDS, an anionic detergent which denatures secondary and non–disulfide–linked tertiary structures, and applies a negative charge to each protein in proportion to its mass.Without SDS, different proteins with similar molecular weights would migrate differently due to differences in mass charge ratio, as each protein has an isoelectric point and molecular weight particular to its primary structure. This is known as Native PAGE. Adding SDS solves this problem, as it binds to and unfolds the protein, giving a near uniform negative charge along the length of the polypeptide.SDS binds in a ratio of approximately 1.4 g SDS per 1.0 g protein ,giving an approximately uniform mass:charge ratio for most proteins, so that the distance of migration through the gel can be assumed to be directly related to only the size of the protein.

SDS PAGE

Chemical ingredients and their roles

Polyacrylamide gel (PAG) had been known as a potential embedding medium for sectioning tissues as early as 1954. Two independent groups, Davis and Raymond, employed PAG in electrophoresis in 1959. It possesses several electrophoretically desirable features that make it a versatile medium. PAGE separates protein molecules according to both size and charge. It is a synthetic gel, thermo-stable, transparent, strong, relatively chemically inert, can be prepared with a wide range of average pore sizes . The pore size of a gel is determined by two factors, the total amount of acrylamide present (%T) (T = Total acrylamide-bisacrylamide monomer concentration) and the amount of cross-linker (%C) (C = Crosslinker concentration).

Pore size decreases with increasing %T; with cross-linking, 5%C gives the smallest pore size. Any increase or decrease in %C increases the pore size, as pore size with respect to %C is a parabolic function with vertex as 5%C. This appears to be because of nonhomogeneous bundling of strands in the gel.This gel material can also withstand high voltage gradients, feasible for various staining and destaining procedures, and can be digested to extract separated fractions or dried for autoradiography and permanent recording. There are two layers of gel, namely stacking or spacer gel, and resolving or separating gel.

Stacking gel

The stacking gel is a large pore PAG (4%T). This gel is prepared with Tris/HCl buffer pH 6.8 of about 2 pH units lower than that of electrophoresis buffer (Tris/Glycine). These conditions provide an environment for Kohlrausch reactions determining molar conductivity, as a result, SDS-coated proteins are concentrated to several fold and a thin starting zone of the order of 19 μm is achieved in a few minutes. This gel is cast over the resolving gel. The height of the stacking gel region is always maintained more than double the height and the volume of the sample to be applied. This is based on isotachophoresis.

Chemical ingredients

Tris (tris (hydroxy methyl) aminomethane) (C4H11NO3; mW: 121.14). It has been used as a buffer because it is an innocuous substance to most proteins. Its pKa is 8.3 at 20 °C, making it a very satisfactory buffer in the pH range from roughly 7 to 9. Glycine (Amino Acetic Acid) (C2H5NO2; mW: 75.07). Glycine has been used as the source of trailing ion or slow ion because its pKa is 9.69 and mobility of glycinate are such that the effective mobility can be set at a value below that of the slowest known proteins of net negative charge in the pH range. The minimum pH of this range is approximately 8.0.

Acrylamide (C3H5NO; mW: 71.08). It is a white crystalline powder. While dissolving in water, autopolymerisation of acrylamide takes place. It is a slow spontaneous process by which acrylamide molecules join together by head on tail fashion. But in presence of free radicals generating system, acrylamide monomers are activated into a free-radical state. These activated monomers polymerise quickly and form long chain polymers. This kind of reaction is known as Vinyl addition polymerisation. A solution of these polymer chains becomes viscous but does not form a gel, because the chains simply slide over one another. Gel formation requires hooking various chains together. Acrylamide is a neurotoxin. It is also essential to store acrylamide in a cool dark and dry place to reduce autopolymerisation and hydrolysis.

Bisacrylamide (N,N'-Methylenebisacrylamide) (C7H10N2O2; mW: 154.17).

Bisacrylamide is the most frequently used cross linking agent for poly acrylamide gels. Chemically it is thought of having two-acrylamide molecules coupled head to head at their non-reactive ends.

Sodium Dodecyl Sulfate (SDS) (C12H25NaO4S; mW: 288.38). SDS is the most common dissociating agent used to denature native proteins to individual polypeptides. When a protein mixture is heated to 100 °C in presence of SDS, the detergent wraps around the polypeptide backbone. It binds to polypeptides in a constant weight ratio of 1.4 g/g of polypeptide. In this process, the intrinsic charges of polypeptides becomes negligible when compared to the negative charges contributed by SDS. Thus polypeptides after treatment becomes a rod like structure possessing a uniform charge density, that is same net negative charge per unit length.

Ammonium persulfate (APS) (N2H8S2O8; mW: 228.2). APS is an initiator for gel formation.

TEMED (N, N, N', N'-tetramethylethylenediamine) (C6H16N2; mW: 116.21). Chemical polymerisation of acrylamide gel is used for SDS-PAGE. It can be initiated by ammonium persulfate and the quaternary amine, N,N,N',N'-tetramethylethylenediamine (TEMED). The rate of polymerisation and the properties of the resulting gel depends on the concentration of APS and TEMED. The lowest catalytic concentrations that will allow polymerisation in the optimal period of time should be used. APS and TEMED are used, approximately in equimolar concentrations in the range of 1 to 10 mM.

Chemicals for processing and visualization

Bromophenol blue (BPB) (3',3",5',5" tetrabromophenolsulfonphthalein) (C19H10Br4O5S; mW: 669.99). BPB is the universal marker dye. Proteins and nucleic acids are mostly colourless. When they are subjected to electrophoresis, it is important to stop the run before they run off the gel. BPB is the most commonly employed tracking dye, because it is viable in alkali and neutral pH, it is a small molecule, it is ionisable and it is negatively charged above pH 4.6 and hence moves towards the anode. Being a small molecule it moves ahead of most proteins and nucleic acids. As it reaches the anodic end of the electrophoresis medium electrophoresis is stopped. It can bind with proteins weakly and give blue colour.

Glycerol (C3H8O3; mW: 92.09). (C3H8O3; mW: 92.09). It is a preservative and a weighing agent. Addition of glycerol It is a preservative and a weighing agent. Addition of glycerol (20-30 or 50%) is often recommended for the storage of (20-30 or 50%) is often recommended for the storage of enzymes. Glycerol maintains the protein solution at very low enzymes. Glycerol maintains the protein solution at very low temperature, without freezing. It also helps to weigh down the temperature, without freezing. It also helps to weigh down the sample into the wells without being spread while loading. sample into the wells without being spread while loading. Coomassie Brilliant Blue R-250 (CBB)(C45H44N3NaO7S2; mW: R-250 (CBB)(C45H44N3NaO7S2; mW: 825.97). 825.97). CBB is the most popular protein stain. It is an anionic CBB is the most popular protein stain. It is an anionic dye, which binds with proteins non-specifically. The structure dye, which binds with proteins non-specifically. The structure of CBB is predominantly non-polar. So is usually used of CBB is predominantly non-polar. So is usually used (0.025%) in methanolic solution (40%) and acetic acid (7%). (0.025%) in methanolic solution (40%) and acetic acid (7%). Proteins in the gel are fixed by acetic acid and simultaneously Proteins in the gel are fixed by acetic acid and simultaneously stained. The excess dye incorporated in the gel can be stained. The excess dye incorporated in the gel can be removed by destaining with the same solution but without the removed by destaining with the same solution but without the dye. The proteins are detected as blue bands on a clear dye. The proteins are detected as blue bands on a clear background. As SDS is also anionic, it may interfere with background. As SDS is also anionic, it may interfere with staining process. Therefore, large volume of staining solution staining process. Therefore, large volume of staining solution is recommended, approximately ten times the volume of the is recommended, approximately ten times the volume of the gel.gel.

Butanol (C4H10O; mW: 74.12). Water saturated butanol is used as an overlay solution on the resolving gel.

2-Mercaptoethanol (HS-CH2CH2OH; mW: 78.13). 2-Mercaptoethanol is a reducing agent used to disrupt disulfide bonds to ensure the protein is fully denatured before loading on the gel; ensuring the protein runs uniformly.

Reducing SDS-PAGEBesides the addition of SDS, proteins may optionally be briefly heated to near boiling in the presence of a reducing agent, such as dithiothreitol (DTT) or 2-mercaptoethanol (beta-mercaptoethanol/BME), which further denatures the proteins by reducing disulfide linkages, thus overcoming some forms of tertiary protein folding, and breaking up quaternary protein structure (oligomeric subunits). This is known as reducing SDS-PAGE, and is most commonly used. Non-reducing SDS-PAGE (no boiling and no reducing agent) may be used when native structure is important in further analysis.

Electrophoresis and staining The denatured proteins are subsequently applied to one end of a layer of polyacrylamide gel submerged in a suitable buffer. An electric current is applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the anode. Depending on their size, each protein will move differently through the gel matrix: short proteins will more easily fit through the pores in the gel, while larger ones will have more difficulty (they encounter more resistance). After a set amount of time (usually a few hours- though this depends on the voltage applied across the gel; higher voltages run faster but tend to produce somewhat poorer resolution), the proteins will have differentially migrated based on their size; smaller proteins will have traveled farther down the gel, while larger ones will have remained closer to the point of origin .

Therefore, proteins may be separated roughly according to size . Following electrophoresis, the gel may be stained with Coomassie Brilliant Blue R-250 or silver stain), allowing visualisation of the separated proteins, or processed further (e.g. Western blot). After staining, different proteins will appear as distinct bands within the gel. It is common to run molecular markers of known molecular weight in a separate lane in the gel, in order to calibrate the gel and determine the weight of unknown proteins by comparing the distance traveled relative to the marker. The acrylamide concentration of the gel can also be varied, generally in the range from 5% to 25%. Lower percentage gels are better for resolving very high molecular weight proteins, while much higher percentages are needed to resolve smaller proteins.

Gel electrophoresis is usually the first choice as an assay of protein purity due to its reliability and ease. The presence of SDS and the denaturing step causes proteins to be separated solely based on size. False negatives and positives are possible. A comigrating contaminant can appear as the same band as the desired protein. This comigration could also cause a protein to run at a different position or to not be able to penetrate the gel. This is why it is important to stain the entire gel including the stacking section. Coomassie Brilliant Blue will also bind with less affinity to glycoproteins and fibrous proteins, which interferes with quantification.

Cell 2004

Silver staining

Classical Coomassie Brilliant Blue staining can usually detect a 50 ng

protein band, Silver staining increases the sensitivity typically

50 times. Many variables can influence the colour intensity and every protein has its own staining characteristics; clean glassware,

pure reagents and water of highest purity are the key points to

successful staining.

Buffer systemsPostulated migration of proteins in a Laemmli gel system A: Stacking gel, B: Resolving gel, o: sample application c: discontinuities in the buffer and electrophoretic matrixMost protein separations are performed using a "discontinuous" buffer system that significantly enhances the sharpness of the bands within the gel. During electrophoresis in a discontinuous gel system, an ion gradient is formed in the early stage of electrophoresis that causes all of the proteins to focus into a single sharp band. This occurs in a region of the gel that has larger pores so that the gel matrix does not retard the migration during the focusing or "stacking" event. Negative ions from the buffer in the tank then "outrun" the SDS-covered protein "stack" and eliminate the ion gradient so that the proteins subsequently separate by the sieving action in the lower, "resolving" region of the gel.

Many people continue to use a tris-glycine or "Laemmli" buffering system that stacks at a pH of 6.8 and resolves at a pH of ~8.3-9.0. These pHs promote disulfide bond formation between cysteine residues in the proteins, especially when they are present at high concentrations because the pKa of cysteine ranges from 8-9 and because reducing agent present in the loading buffer doesn't co-migrate with the proteins. Recent advances in buffering technology alleviate this problem by resolving the proteins at a pH well below the pKa of cysteine (e.g., bis-tris, pH 6.5) and include reducing agents (e.g. sodium bisulfite) that move into the gel ahead of the proteins to maintain a reducing environment. An additional benefit of using buffers with lower pHs is that the acrylamide gel is more stable so the gels can be stored for long periods of time before use.

Two-dimensional gel electrophoresis

Basis for separation

2-D electrophoresis begins with 1-D electrophoresis but then separates the molecules by a second property in a direction 90 degrees from the first. In 1-D electrophoresis, proteins (or other molecules) are separated in one dimension, so that all the proteins/molecules will lie along a lane but be separated from each other by a property (e.g. isoelectric point). The result is that the molecules are spread out across a 2-D gel. Because it is unlikely that two molecules will be similar in two distinct properties, molecules are more effectively separated in 2-D electrophoresis than in 1-D electrophoresis.The two dimensions that proteins are separated into using this technique can be isoelectric point, protein complex mass in the native state, and protein mass.

To separate the proteins by isoelectric point is called isoelectric focusing (IEF). Thereby, a gradient of pH is applied to a gel and an electric potential is applied across the gel, making one end more positive than the other. At all pHs other than their isoelectric point, proteins will be charged. If they are positively charged, they will be pulled towards the more negative end of the gel and if they are negatively charged they will be pulled to the more positive end of the gel. The proteins applied in the first dimension will move along the gel and will accumulate at their isoelectric point; that is, the point at which the overall charge on the protein is 0 (a neutral charge).

In native polyacrylamide gel electrophoresis (native PAGE), proteins remain in their native state and are separated in the electric field following their mass and the mass of their complexes respectively. To obtain a separation by size and not by net charge, as in IEF, an additional charge is transferred to the proteins by the use of coomassie or lithium dodecyl sulfate (LDS). After completion of the first dimension the complexes are destroyed by applying the denaturing SDS-PAGE in the second dimension, where the penchod proteins of which the complexes are composed of are separated by their mass.Before separating the proteins by mass, they are treated with sodium dodecyl sulfate (SDS) along with other reagents (SDS-PAGE in 1-D). This denatures the proteins (that is, it unfolds them into long, straight molecules) and binds a number of SDS molecules roughly proportional to the protein's length.

Because a protein's length (when unfolded) is roughly proportional to its mass, this is equivalent to saying that it attaches a number of SDS molecules roughly proportional to the protein's mass. Since the SDS molecules are negatively charged, the result of this is that all of the proteins will have approximately the same mass-to-charge ratio as each other. In addition, proteins will not migrate when they have no charge (a result of the isoelectric focusing step) therefore the coating of the protein in SDS (negatively charged) allows migration of the proteins in the second dimension (NB SDS is not compatible for use in the first dimension as it is charged and a nonionic or zwitterionic detergent needs to be used). In the second dimension, an electric potential is again applied, but at a 90 degree angle from the first field. The proteins will be attracted to the more positive side of the gel proportionally to their mass-to-charge ratio.

The gel therefore acts like a molecular sieve when the current is applied, separating the proteins on the basis of their molecular weight with larger proteins being retained higher in the gel and smaller proteins being able to pass through the sieve and reach lower regions of the gel.The result of this is a gel with proteins spread out on its surface. These proteins can then be detected by a variety of means, but the most commonly used stains are silver and coomassie staining. In this case, a silver colloid is applied to the gel. The silver binds to cysteine groups within the protein. The silver is darkened by exposure to ultra-violet light. The darkness of the silver can be related to the amount of silver and therefore the amount of protein at a given location on the gel. This measurement can only give approximate amounts, but is adequate for most purposes.

Molecules other than proteins can be separated by 2D electrophoresis. In supercoiling assays, coiled DNA is separated in the first dimension and denatured by a DNA intercalator (such as ethidium bromide or the less carcinogenic chloroquine) in the second. This is comparable to the combination of native PAGE /SDS-PAGE in protein separation.In summary 2D provides resolution according to two traits, whereof one is most often molecular charge. The investigated molecule needs not be protein

Electrophoresis is performed in a matrix with a pH gradient (it is generated by equilibrating a mixture of ampholytes, which under an electric field distribute proportionally to their net charge).

Analysis of proteins by electrophoresis to determine pI of proteins Isoelectric focusing

Analysis of proteins by two dimensional gel electrophoresis 1stdimension ->Isoelectricfocusing

Analysis of proteins by two dimensional gel electrophoresis 1stdimension -

>Isoelectricfocusing2nddimension -> SDS-PAGE

Analysis of proteins by two dimensional gel electrophoresis 1stdimension -

>Isoelectricfocusing2nddimension -> SDS-PAGESeparates by both pI and Mw. It is used to

analyze complex mixtures of proteins

• For 10 ml• 1M Tris-Hcl pH-8.0 500ul• 5M Nacl 300ul• 10% NP40 500ul• 1M NaF 1ml• 250mM Na-vanadate 40ul (add fresh)• 0.1M PMSF 80ul (add fresh)• Protease Cocktail 400ul (add fresh)• Make up the Volume with H2O

Lysis buffer

Separating Gel Preparation: 2 Gels

% Acrylamide 7.5% 10% 12% 15%

H20 9.6 ml 8.0 ml 6.6 ml 4.6ml

1.5 M Tris pH8.8 5.0 ml 5.0 ml 5.0 ml 5.0 ml

10%SDS 200ul 200ul 200ul 200ul

Acrylamide/Bis 5.0 ml 6.7 ml 8.0 ml 10 ml

10%APS 200ul 200ul 200ul 200ul

TEMED 20ul 20ul 20ul 20ul

Stacking Gel: 2 Gels

• H20 7.4 ml• 0.5 M Tris pH 6.8 1.25ml• 10%SDS 100ul• Acrylamide/Bis 1.3 ml• 10% APS 50ul• TEMED 10ul

6X SDS Dye

• 6ml 20% SDS• 4 ml Glycerol• 3.6 ml Tris pH 6.8• 5% BME ( 700ul)• Add a pinch of Bromophenol Blue

10 X SDS –PAGE Electrophoresis running buffer (Make up the volume = 1L with d H20)

• Sl. No. Chemicals 1 L (10X)• 1 Tris base (FW 121.1) 30.3 g• 2 Glycine (FW 75.07) 144 g• 3 SDS 10 g

10X Transfer Buffer

• For 1 litre:• Tris base 29gm• Glycine 145 gm

10X TBS

• For 500ml• Tris base 12.11 gm• Nacl 40 gm

Stripping Buffer

• For 500 ml• 31.25 ml Tris Hcl PH 6.8• 100 ml 10% SDS• Adjust the Vol with H20 • Add BME 5 ul /ml before use

• Prepare the gel with above mentioned compositions.• Before using the cassette, carefully remove the

comb. With a razor blade, remove all pieces of gel that may obstruct the entry of the gel.

• Load your sample with a loading tip to the bottom of the gel. Fill the tank with the Electrophoresis buffer.

• Connect the power pack to the apparatus.• Stain the gel for 45 minutes followed by distaining

• Protein stain reagents: 0.1% Coomassie brilliant blue R250 in 50% methanol, 10% glacial acetic acid. Dissolve the dye in the methanol and water component first, and add the acetic acid. Filter the final solution through Whatman N° 1 paper if necessary.

• Destain reagents: 10% methanol, 7% glacial acetic acid

Western blotting using semi-dry apparatus

• The buffer will range from pH 8.1 to 8.5, depending on the quality of Tris, glycine, dd H2O, and methanol.

• Electroblotting apparatus: Trans-Blot SD semidry cell (Bio-Rad). Whatman paper, 8 sheets (3MM Chr) by gel.

• The size of the blot and Whatman paper have to fit exactly to the size of the separating gel. This point is important to avoid shortcut of current that would not pass through the gel.

• The membrane, Whatman paper, and gel have to be handle with gloves.

Methods After the separation of proteins by SDS-PAGE, the stacking gel

is cut and discarded, the separating gel is briefly rinsed in distilled water 2-3 min and then equilibrated in transfer buffer under gentle agitation during 5-10 min. In the mean time, Whatman paper is soaked in transfer buffer. Place the 5 sheets of Whatman paper pre-wetted on the anode (bottom electrode). Then place the nitrocellulose blot on the stack of Whatman paper. Above, place the gel, conveniently oriented. At this stage, it's relevant to mark with a pencil the blot at the molecular weight marker positions as the stain of the marker tends to disappear with the process. Place 5 other sheets of Whatman paper pre-wetted. Finally, use a clean tube, and press the stack to roll out all air bubbles. Again, take care that all the elements of the stack fit altogether very well, this is the key of a efficient transfer. See Figure bellow.

Mini Trans-

Blot Transfer

Cell

Preparing the Blotting Sandwich

1. Place the cassette with gray sidedown on clean surface

2. Place one pre-wetted fiber pad on the gray side of the cassette

3. Place a sheet of filter paper on the fiber pad

4. Place gel on filter paper taking care to remove air bubbles

5. Place the pre-wetted nitrocellulose membrane on the gel

6. Place the second fiber pad on top

7. Close the cassette firmly DO NOT move gel/filter sandwich

8. Lock the cassette

Prepare for Elect

• Place the closed and locked cassette in the electrode module

• Add the frozen Bio-Ice cooling unit and place in tank

• Fill the tank with buffer

• A stir bar can be added to help maintain the ion and temperature distribution in the tank even

Transfer Proteins from the gel to the nitrocellulose membrane

30 minutes100V

Blotting buffer 1x Tris glycine with 20%

ethanol

Elec

tric

Curre

nt

Use of antibodies as a diagnostic tool

ELISAWestern Blot

• Molecule of interest is injected into primary animal model

• Animal makes antibodies against the molecule

• Antibodies are purified (primary antibody)

• Antibodies from the first animal model are injected into a second animal model

• The second animal produces antibodies against the first antibody (secondary antibody)

Use of antibodies as a diagnostic tool

ELISAWestern Blot

• The secondary antibody is purified and conjugated to a colorimetric substrate or to an enzyme that can cleave a colorimetric compound

• ELISA Western Blot

Western Blot is used to confirm ELISA results

More sensitive

More Specific

Remove membrane from the blotting sandwich and immerse in 25ml of blocking solution for 15minutes

5% non-fat milk: Prevents the primary antibody from binding randomly to the membrane

Phosphate buffered saline (PBS): Provides the correct environment (pH, Salt) to maintain protein shape

0.025% Tween 20: non-ionic detergent that prevents non-specific binding of antibodies to the membrane

Blocking Buffer

Add the Primary Antibody

anti- myosin light-chain

Wash

•Discard blocking solution•Pour 10ml of primary antibody onto the membrane and gently rock for 1 hr at RT or overnight at 4” C with gentle shaking•Primary antibody will bind to the myosin light-chain

•Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer•Add 50ml of wash leave for 10-15 minutes on the rocking platform and repeat the same for 3 times

Add Enzyme-

linked Secondary Antibody

Wash

•Discard wash solution•Pour 10ml of the secondary antibody onto the membrane and gently rock for 30-45 minutes•Secondary antibody will bind to the primary antibody

•Quickly rinse membrane in 50ml of wash buffer and discard the wash buffer•Add 50ml of wash leave for 10 minutes on the rocking platform and repeat the same for three times

Add Enzyme Substrate

Watch for Color Development

•Discard wash solution

•Add 10ml of the enzyme substrate (HRP color detection reagent) onto the membrane

•Incubate for 10 minutes

•The colorimetric substrate is cleaved by the enzyme conjugated (attached) to the secondary antibody

Blot from unstained Gel Blot from stained Gel

• Add some milliliters of transfer buffer on the top of the stack, wet the cathode with a wet paper towel imbibed with transfer buffer. This procedure should not exceed 15 min, because proteins diffuse within the gel and you could lose resolution by being to slow in your handling.

• Connect the blotting unit to a power supply.• At the end, cut the power, remove the blot, and dry it

on a clean aluminum foil with a hair dryer. This helps to fix the protein on the blot. Do not overdry.

• Proceed to Western-blot treatment (see following section). (You can store the blot at –20 °C after soaking in blocking buffer (TBS, 1% milk) for one hour).

Western-blot • After transfer the gel can be stained in Coomassie to

ensure the completion of transfer.• The nitrocellulose membrane is blocked in

TBS/tween/milk (25 mM Tris-HCl, pH 7.5, 150 mM  NaCl, 0,2% (v/v) Tween 20, 1% (w/v) non-fat dried milk) for 1h at RT with gentle rocking.

• Then, incubate the membrane 2h at RT with the primary antibodies (prepared in TBS/tween/milk) and gentle rocking. (This can be done O/N for your convenience)

• Wash 5 times 5 min in TBS/tween/milk (25-50 ml) at RT, gentle rocking.

Western Blot• Then, incubate the membrane 1h at RT with the

secondary antibodies (prepared in TBS/tween/milk) and gentle rocking.

• Wash 5 times 5 min in TBS/tween/milk (25-50 ml) at RT, gentle rocking.

• For alkaline phosphatase staining, incubate the membrane in its substrate BCIP/NPT. 15 min of incubation is an average time that you may modify accordingly to your result.

• Wash thoroughly with water to stop the reaction. Dry the blot with a hair dryer (do not over-dry). Store the membrane in the dark for example between Whatman sheets in your lab-book (formazan staining is light sensitive).