Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis

(SDS PAGE) Analysis of Proteins

The principleWhen proteins are separated by electrophoresis through a gel matrix, smaller proteins migrate faster due to less resistance from the gel matrix. Other influences on the rate of migration through the gel matrix include the structure and charge of the proteins.

In SDS-PAGE, the use of sodium dodecyl sulfate (SDS) and polyacrylamide gel largely eliminates the influence of the structure and charge, and proteins are separated solely based on polypeptide chain length.

SDS is a detergent with a strong protein-denaturing effect and binds to the protein backbone at a constant molar ratio. In the presence of SDS and a reducing agent that cleaves disulfide bonds critical for proper folding, proteins unfold into linear chains with negative charge proportional to the polypeptide chain length

Polymerized acrylamide (polyacrylamide) forms a mesh-like matrix suitable for the separation of proteins of typical size. The strength of the gel allows easy handling. Polyacrylamide gel electrophoresis of SDS-treated proteins allows researchers to separate proteins based on their length in:

1. an easy

2. inexpensive

3. relatively accurate manner.

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A polyacrylamide gel is prepared by co-polymerising acrylamide and methylene bis-acrylamide; the reaction is initiated by a source of radicals (typically ammonium persulphate, but sometimes riboflavin and uvirradiation) and the catalyst tetramethyleneethylene diamine (TEMED).

Methylene bis-acrylamide forms cross-links between the chains of polyacrylamide.

The percentage of methylene bis-acrylamide in the mixture, and hence the extent of cross-linking, determines the size of the pores in the gel, and hence the extent to which proteins of different size are retarded in the electric field.

Polymerization of acrylamide and bisacrylamide monomers is induced by ammonium persulfate (APS), which spontaneously decomposes to form free radicals. TEMED, a free radical stabilizer, is generally included to promote polymerization.

Materials

• PAGE Rigs including glass plates (10 x 20 cm), spacers, comb, and clamps

• Power supply• Protein sample• Bio-Rad Laemmli Sample Buffer (contains SDS and either sucrose or

glycerol)• 2-Mercaptoethanol (reduces disulfide bonds, disrupts protein cross-

links)• MW Markers (already prepared in sample buffer)

Gel Cassette Assembly (Bio-Rad Mini Protean 3)1. Clean and completely dry the glass plates, combs, and any other

pertinent materials.2. Place a short plate on top of a spacer plate. Insert both plates into

the green casting frame on a flat surface. Be sure that the "legs" of the casting frame are down. Clamp the casting frame and check that the plates are level on the bottom.

3. Put the casting frame into the casting stand.

Preparation of the Gel

• Combine all reagents except the TEMED for the 14% separating gel.14% Separating Gel Components (4.195 mL)• 1.184 milliliters deionized water• 1.96 milliliters 30% acrylamide/Bis (Warning: Acrylamide is a

neurotoxin. Use gloves, do not ingest.)• 1.0 milliliters 1.5 M Tris, pH 8.8• 21 microliters 20% SDS• 12 microliters 10% ammonium persulfate

18 microliters TEMED, pH 8.9

• When ready to pour the gel, quickly add the TEMED, mix using a Pasteur pipette, and transfer the separating gel solution between the glass plates in the casting chamber to about 3/4 inch below the short plate.

• A small layer of butanol can be added on top of the gel prior to polymerization to straighten the level of the gel and remove unwanted air bubbles that may be present. Butanol will not mix with the aqueous separating gel solution. Once the gel has polymerized, the butanol can be removed by absorption with Kim wipes or filter paper. Rinse the top layer of the gel with dI water prior to pouring the stacking gel.

• Insert the well forming comb into the opening between the glass plates.

• Combine all reagents except the TEMED for the 5% stacking gel.

• 5.1% Stacking Gel (2.484 mL)• 0.4 milliliters deionized water• 1.8 milliliters 30% acrylamide/Bis• 0.25 milliliters 0.5 M Tris, pH 6.8• 12 microliters 20% SDS• 17 microliters 10% ammonium persulfate• 5 microliters TEMED• When ready to pour the gel, quickly add the TEMED, mix using a Pasteur pipette, and transfer the stacking

gel solution between the glass plates in the casting chamber.

• Both the separating and stacking gels should polymerize within six minutes.

• Once the stacking gel has polymerized, the comb can be gently removed. The polymerized gel between the short plate and spacer plate forms the "gel cassette".

Sample Preparation

• Place some water in a 600 mL beaker and leave on a hot plate to boil. (This can take 15 minutes or more.)

• add 50 mL of 2-mercaptoethanol to 950 mL of Laemmli sample buffer.

• Combine 10 mL of each protein sample with 20 mL of Laemmli sample buffer plus 2-mercaptoethanol in microcentrifuge tubes.

• In separate tubes, aliquot 10 mL of MW marker. (MW markers are already prepared in Laemmlisample buffer.)

• Boil the samples for no more than 5 minutes to fully denature the proteins. Leave the samples at room temperature until ready to load onto the gel.

Electrophoresis• Remove the gel cassette from the casting stand and place it in the electrode assembly with the short plate on the inside.

• Slide the electrode assembly (with the gel cassette) into the clamping frame. Press down on the electrode assembly while clamping the frame to secure the electrode assembly. This step is important to minimize potential leakage during the electrophoresis experiment.

• Pour some 1X electrophoresis buffer into the opening of the casting frame between the gel cassettes. Add enough buffer to fill the wells of the gel. Use a gel loading tip to pipette some buffer into each well to ensure cleanliness.

• When all wells are sufficiently cleaned, slowly pipette no more than 30 mL of denatured sample or MW marker into each well. A yellow guide can be placed on top of the electrode assembly to aid in loading the gel.

• When the gel has been loaded, lower the clamping frame into the electrophoresis tank.

• Fill the region outside of the frame with 1X electrophoresis buffer.

• Cover the tank with the lid aligning the electrodes (black or red) appropriately.

• Connect the electrophoresis tank to the power supply.

• Allow the samples to run at 30 mA until the dye front reaches the bottom of the gel. This can take as long as 1 hour.

• When electrophoresis is complete, turn off the power supply and disassemble the apparatus.

Summary of SDS- PAGE procedure: • Sample preparation: Samples may be any material containing proteins

• Mixing with SDS: The sample is mixed with SDS, anionic detergent. To denatures secondary and non–disulfide–linked tertiary structures. To apply a negative charge to each protein.

• Heating: The samples are heated at 60°C. To promote protein denaturation, helping SDS to bind.

• Addition of tracking dye: A tracking dye may be added to the protein solution. As it has a higher electrophoretic mobility which allow the experimenter to track the progress of the protein solution through the gel.

• Preparing acrylamide gels: Gels are usually polymerized between two glass plates in a gel caster, with a comb inserted at the top to create the sample wells. After the gel is polymerized the comb can be removed and the gel is ready for electrophoresis

• Electrophoresis: Various buffer systems are used in SDS-PAGE depending on the nature of the sample and the experimental objective. An electric field is applied across the gel, causing the negatively-charged proteins to migrate across the gel towards the positive (+) electrode (anode)

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