Immunology

Experiment 7

Aim of the Experiment

To learn the technique of SDS- Polyacrylamide Gel Electrophoresis

Introduction

SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel electrophoresis, is a technique used in biochemistry, genetics and molecular biology to separate proteins according to their molecular weight. The electrophoretic mobility of proteins depends upon their size. The purpose of SDS-PAGE is to separate proteins according to their size. As proteins are amphoteric compounds, their net charge can therefore be determined by the pH of the medium in which they are suspended. Therefore, at a given pH and under non-denaturing conditions, the electrophoretic separation of proteins is determined by both size and charge of molecules. As proteins are high molecular weight molecules, it needs porous gels to get separated. Polyacrylamide gels are those which provide a means of separating proteins by size as they are porous. This kit enables the students to learn the technique of SDS-PAGE.

Principle

To separate different protein molecules of different shapes and sizes, they first have to be denatured so that the proteins no longer have any secondary, tertiary or quaternary structure. Sodium dodecyl sulphate (SDS) is an anionic detergent which denatures proteins by “wrapping around” the polypeptide backbone. SDS denatures all the proteins to their respective primary structure. SDS confers a negative charge to the polypeptide in proportion to its length.

SDS treatment has two important features:

1. All proteins retain only their primary structure.

2. All proteins have a large amount of negative charge.

Polyacrylamide is the best gel recommended to provide such an environment. Polyacrylamide is a synthetic gel which is thermo-stable, transparent, strong and relatively chemically inert and can be prepared with a wide range of average pore sizes. It can withstand high voltage gradients and is feasible to various staining and destaining procedures and can be digested to extract separated fractions or dried for autoradiography and permanent recording. A polymer gel is formed of acrylamide monomers and the proteins are run through this gel by electrophoresis, hence this entire process is called Polyacrylamide Gel Electrophoresis (PAGE).

There are two layers of gel, namely Stacking or spacer gel, and Separating or resolving gel.

Stacking gel - The stacking gel contains large pores of polyacrylamide gel (generally 5%). This gel is prepared with Tris buffer of pH 6.8 which is of about 2 pH units lower than that of the electrophoresis buffer. This gel is formed over the separating gel.

Separating Gel - The separating gel contains small pores of polyacrylamide gel (5-30%). The Tris buffer used is of pH 8.8. In this gel, macro molecules separate according to their size.

The materials used in SDS-PAGE and their roles are as follows:

1. Tris: It is used as a buffer because it is an innocuous substance to most proteins. Its pKa is 8.3 at 20oC and reasonably a very satisfactory buffer in the pH range 7.0 – 9.0

2. Acrylamide: This is a white crystalline powder and while dissolving in water, autopolymerisation 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 of polymers. This kind of reaction is known as Vinyladdition polymerisation.

3. Bisacrylamide (N,N’-Methylenebisacrylamide): Bisacrylamide is the most frequently used cross linking agent for polyacrylamide gels. Chemically it has two acrylamide molecules coupled head to head fashion at their non-reactive ends.

4. Sodium Dodecyl Sulphate (SDS): SDS is the most common denaturing 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 become negligible when compared to the negative charges contributed by SDS. Thus, polypeptides after treatment become a rod like structure possessing a uniform charge density, that is same net negative charge per unit length.

5. Ammonium Persulphate (APS): APS is an initiator for gel formation.

6. N, N, N’, N’-tetramethylethylenediamine (TEMED): 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).

Instructions

1. Read the entire procedure carefully before starting the experiment.

2. Preparation of 10% APS Solution: Before starting the experiment, dissolve 0.2 g of Ammonium persulphate in distilled water to make a final volume of 2.0 ml. Store at 2-8oC. Use within 3 months.

3. Preparation of 1X Tris-Glycine-SDS Gel Running Buffer: To prepare 500 ml of 1X Tris-Glycine-SDS Gel Running Buffer, take 100 ml of 5X Tris-Glycine-SDS Gel Running Buffer and add 400 ml sterile distilled water*. Store at 2-8 C. Mix well before use. The 1X Tris-Glycine-SDS Gel Running Buffer can be reused 4-5 times.

4. Thaw all refrigerated samples before use.

5. Clean the entire apparatus with detergent and then with distilled water*. Ensure that the plates are free of detergent.

Note: Acrylamide is a potential neurotoxin and should be treated with great care. Always wear a face mask and use gloves.

* Molecular biology grade water is recommended

Materials required

• Glass wares: Conical flask, Measuring cylinder, Beaker

• Reagents: Distilled water

• Other requirements: Protein Electrophoresis apparatus, Micropipettes, Tips, Microwave/Burner/Hotplate,

• Pour the gel in-between the plates and allow it to solidify for an hour. Immediately after the gel is poured, add distilled water to level the gel.

• After an hour pour off the water by inverting the casting assembly.

• After addition of TEMED gently mix all the components by swirling the beaker. Pour the stacking gel on top of the separating gel and immediately place the comb avoiding air bubbles. Allow it to solidify for 30 minutes.

• Remove the gel from Staining Solution. The Staining Solution can be re-used 2-3 times.

• Wash the gel by rinsing in distilled water till a considerable amount of stain leaches out from the gel. Keep changing the distilled water for 3-4 times.

• Add 50 ml of Destaining Solution to the gel. Destaining should be carried out with constant moderate shaking.

• Continue destaining till clear, distinct bands are observed.

• Remove the gel from Destaining Solution. The Destaining Solution can be re-used 2-3 times.

Interpretation

After staining and destaining the gel compare the molecular weight of the samples with that of the protein marker. Protein sample 1 contains serum hence multiple bands can be seen. Protein Sample 2 is a purified protein hence 1 major band can be seen.

Reference Material

• Running a protein gel

• SDS-PAGE

• Himedia Protocol

Questions

Q.1. Why did you use polyacrylamide gels to analyze your protein fractions rather than agarose gels?

Q.2 What is the difference between Stacking gel and Resolving gel?

Q.3 How can you speed up the electrophoresis?

Q.4 What are the safety considerations during electrophoresis?