SDS PAGE Protocol

What is SDS PAGE?

The full form of SDS PAGE is sodium dodecyl sulphate – polyacrylamide gel electrophoresis. It was developed by biochemistry professor Ulrich K. Laemmli. It is a powerful, well-established and quite an elaborate electrophoresis technique used to obtain high-resolution analytical separation of protein aggregates and fragments. SDS PAGE is an easy, advanced, inexpensive technique which provides accurate results within a decent time limit. It is highly preferred by research professionals for detailed studies of proteins.

Generally, the basic electrophoresis separates the mixture of molecules based on their molecular weight. Similarly, the SDS PAGE works on the migration of charged protein fragments under the influence of an electric field.

Content: SDS PAGE

1. SDS PAGE Requirements
2. Preparation of Reagents
3. SDS PAGE Protocol
4. SDS PAGE Electrophoresis
5. Principle of SDS PAGE
6. SDS PAGE Gel
7. What is the role of SDS in SDS PAGE?
8. SDS PAGE Results
9. Applications of SDS PAGE
10. Limitations of SDS PAGE

SDS PAGE Requirements

• Gel casting apparatus and electrophoresis equipment
• 30% acrylamide/ 0.8% Bis-acrylamide
  Note: It is a potent neurotoxin and can be absorbed by the skin. One should take appropriate measures while using it. It is an inert material which does not react with the protein sample.
• 2.5x separating gel buffer
• 5x stacking gel buffer
• TEMED
• 10% APS
• Butanol
• 5x electrophoresis buffer
• 5x SDS-PAGE sample buffer
• Protein samples

Preparation of Reagents

Acrylamide and bis-acrylamide stock solution

• 30% acrylamide – 300g
• 0.8% bis-acrylamide – 8g

2.5x separating gel buffer

• 1.875 M Tris Cl – 227.1g
• 0.25% SDS, pH 8.9 – 2.5g

Note: Adjust pH to 8.9 using HCL

5x stacking gel buffer

• 0.3 M Tris-phosphate – 36.3g
• 0.5% SDS, pH 6.7 – 5g

Note: Adjust pH to 6.7 using phosphoric acid

5x electrophoresis buffer

• 0.5M Tris base – 60.6g
• 1.92M Glycine – 144.1g
• 0.5% SDS – 5g

Note: pH should be 8.8 without adjustment.

SDS PAGE Protocol

1. Assemble the gel casting plates vertically using spacers. The plates must be cleaned and dried thoroughly before use.

2. Mark the level with a marker upto where the gel is to be poured (a few millimetres below the comb).

3. Or making the resolving buffer add the following components to the beaker:

• 2.2 ml 30% acrylamide /0.8% Bisacrylamide stock solution
• 2.2 ml 2.5x resolving gel buffer
• 1.1 ml distilled water
• 5µl TEMED

Note

• The volume of the above components may change according to the protein sample to be separated.
• The required volume will vary as per the size of the gel casting apparatus.

4. Just before pouring the resolving gel solution into the caster, add 50µl of 10% APS and mix well.

Note

• The prepared gel should instantly be poured before the polymerises begins.
• Every time one should prepare a fresh APS solution; otherwise, its efficiency might degrade.

5. Now pour the gel into the assembled caster plates up to the marked level.

6. Let the gel polymerise (it takes around 20 min generally). Once polymerised, pour it off with butanol or double distilled water. The remaining liquid on the gel can be removed with the help of filter paper.

Note

The butanol can harm the casting apparatus, therefore distilled water.

7. Now, for preparing the stacking gel solution, add the following components to a beaker:

• 0.28 ml 30% acrylamide/Bisacrylamide stock solution
• 0.33ml 5x stacking gel buffer
• 1ml double distilled water
• 2µl TEMED

8. After preparing the stacking gel solution, add 15µl of 10% APS just before pouring and mixing well.

9. Now, pour the stacking gel on top of the resolving gel carefully.

10. Once the gel is poured, insert the comb to avoid air bubbles.

11. Let it polymerise (it takes around 10 min). Later place the gel in the electrophoresis chamber and fill it with electrophoresis buffer.

12. Now, slowly remove the combs, and you can see the wells.

13. Add 2µl of sample buffer to 8µl of protein which makes the final volume as 10ul (adding 1 volume of sample buffer to 4 volumes of protein sample).

14. vortex the sample briefly and heat them for 5 min at 95^oC.

Note

Open the lids or pierce a hole with a needle in the lid of the sample tube to release the pressure generated due to heating.

SDS PAGE Electrophoresis

15. Load the samples and run the gel. Ideally, the electric field is applied for 100-150 volts for 40-60 minutes (Or until the bromophenol blue dye travels just above the bottom of the gel.)

Note

• Ensure the electrode are attached at their respective right ends.
• One should keep a constant look at the running gel because if the gel runs for too long, there are chances that you may lose the protein bands to the buffer.

Principle of SDS PAGE

SDS PAGE separates the proteins based on their size and molecular weight when kept under the electric field. The sample is heated under denaturing and reducing conditions so as to unfold the proteins. This is done by using SDS detergent, which coats the unfolded protein fragments and gives them a uniform net negative charge.

As the prepared sample is loaded in the gel matrix and the electric field is applied, the negatively charged protein fragments migrate from the negative end towards the positive electrode. This way, they are segregated on the acrylamide mesh by the molecular sieving effect.

SDS PAGE gel

Two types of polyacrylamide gel are used here- stacking gel and resolving gel. Both gels contain acrylamide and bis-acrylamide, which are polymerized to generate polyacrylamide. But their concentration varies in resolving and stacking gel.

Stacking gel

The stacking gel is in the upper layer and has a low concentration of acrylamide. Thus, the mesh size here is large and allows the fragments to pass easily. The pH of Stacking gel is 6.8, which dissociates only a few glycine molecules. The Cl^– ions from tris HCL move quickly and ahead of glycine.

Here, the SDS-treated protein sample is sandwiched between glycine on the upper side and Cl^– ion on the lower side. Thereby compressing the protein into bands that are much smaller than the initially loaded volume. The main purpose of this gel is to transfer all protein fragments to the resolving gel at the same time.

Resolving gel

Whereas the resolving gel is in the lower layer and has a higher concentration of acrylamide. Thus, the mesh is very fine and allows the protein molecules to move slowly through it. The pH of the resolving gel is 8.8, which finely dissociates glycine molecules, increasing the migration speed of protein. In resolving gel, the migration speed of each protein relies on its molecular weight.

Thus, the proteins with smaller weights travel easily while the larger-weight proteins move slowly through the pores. As the electrophoresis ends, the proteins travel to different distances as per their sizes, thereby attaining the purpose of protein separation.

What is the role of SDS in SDS PAGE?

SDS PAGE involves the separation of a mixture of proteins based on their size. But the proteins are large biomolecules formed by peptide linking between the amino acid residues. They exist in one or more spatial conformations. They generate complexly folded tertiary and quaternary structures formed due to the non-covalent interactions like:

• Hydrophobic interactions
• Hydrogen bond
• Ionic bond
• Disulphide bond

Thus, in order to fragmentise the protein, these bonds are broken by using SDS detergent. SDS is an anionic surfactant that bears a net negative charge on it. When the sample is heated with SDS, it denatures the proteins by disturbing the interlinking bonds, including hydrophobic interactions, hydrogen bonds, and ionic bonds.

Whereas beta-mercaptoethanol is used for cleaving the disulphide bridges. Hence the complex protein breaks down into fragments of varied molecular weight. As soon as the protein comes to its primary structure, the SDS molecules cover the entire fragment giving it a net negative charge.

Role of APS

APS stands for Ammonium per sulphate which is an oxidising agent. It spontaneously decomposes to generate free radicals. These free radicles initiate and catalyse the polymerisation of acrylamide and bis-acrylamide.

Role of TEMED

TEMED stands for N, N, N’, N’-Tetramethylethylenediamine. It is a free radical stabiliser which stabilises the radicals released by the APS in order to achieve improved polymerisation.

Note: The APS and TEMED work as catalysts for the polymerisation reaction. Therefore, one should add them last because as soon as they are added, the polymerization reactions start.

SDS PAGE Results

After the protein electrophoresis, you can see the separated bands with your naked eyes. Thus, the gel is stained by using Coomassie brilliant blue dye or silver stain. The staining involves three subsequent processes – fixation, staining and decolourisation. After performing these steps, you can clearly observe the distributed bands.

Applications

• It helps in measuring the molecular weight of the molecules present in a mixture.
• You can approximate the size of the protein by comparing its migration distance with that of the standard.
• Prominently aids the protein studies as it is highly used for estimating protein size, purity of the protein etc.
• Also, it is used in peptide mapping and protein ubiquitination.
• Helpful in comparing the polypeptide composition of different protein structures.
• Also used to separate the HIV proteins in HIV tests.
• Separated fragments are useful for further analytical techniques like western blotting.
• Used to estimate the number and size of the polypeptide subunits.
• Also, it aids the analysis of post-transcriptional modifications.

Limitations

• It is a lengthy and time-consuming process because of so many steps.
• The vertically casted gel is difficult to set. There are very high chances of leakage between the stacking and resolving gel.
• You cannot reuse the gel after the experiments.
• The process involves toxic monomers.

This is all about SDS PAGE.