SDS-PAGE

SDS PAGE, or Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis, is a technique for separating proteins depending on their molecular weight. SDS is an anionic surfactant and detergent. SDS breaks down the non-covalent links of protein molecules. The method of separating protein molecules according to their electrophoretic mobility is frequently used in molecular biology, genetics, forensics, and biotechnology. In this article, we will read about SDS-PAGE, its principles, its methods, and procedures required to carry out its process, and its applications.

Define SDS-PAGE

SDS-PAGE or Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique used to separate proteins based on their molecular weight.

What is Electrophoresis?

A laboratory procedure called electrophoresis is used to separate and examine molecules according to their size and charge, including proteins, DNA, RNA. The process is based on charged particles moving through a medium—usually a gel in an electric field. There are several kinds of electrophoresis, such as DNA, RNA, and protein electrophoresis, each designed for certain macromolecules and applications.

The fundamental idea is to pass an electric current through a gel or other supporting material that holds the relevant sample. Due to their charge, the molecules in the sample migrate across the medium at different speeds based on their size and charge. Larger or fewer negatively charged molecules travel more slowly, whereas smaller and more negatively charged molecules move faster.e.g.: Gel electrophoresis, paper electrophoresis, immunoelectrophoresis, etc.

What is SDS-PAGE?

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis is referred to as SDS-PAGE. Proteins can be separated and analysed using this commonly used laboratory approach according to their size. For characterizing protein samples, determining their molecular weights, and evaluating purity, SDS-PAGE is especially helpful. The approach requires the use of a polyacrylamide gel matrix and the detergent sodium dodecyl sulphate (SDS) to denature and linearize proteins.

Principle of SDS-PAGE

A charged molecule when placed in an electric field migrates to the oppositely charged electrode. The charged molecules get separated based on the relative mobility of charged species. The less electrophoretic resistance in faster migration of smaller molecules. The protein structure and the charge on it also influence the migration rate. SDS and polyacrylamide remove the impact of protein structure and charge. It separates the protein based on the length of the polypeptide chain.

Materials Required

Material used in SDS-PAGE: SDS-PAGE requires samples, gels, staining and destaining buffers or dyes, running buffers, power supplies, and a reference ladder.

Material	Role
Protein/Samples	To check the purity.
Gels	Smooth migration of sample.
Electrophoresis Chambers	For fitting the SDS-PAGE.
Electrophoresis Chambers	Migration of sample in the gel.
Staining and Destaining Buffer	To make the sample visible.
Power Supplies	Convert the AC to DC.
Reference Protein Ladder	To locate the desired protein from the sample.

Procedure of SDS-PAGE

Gel Preparation

• Mix all the reagents (except TEMED).
• Once the gel is ready add TEMED in it.
• Pour the separating gel into the casting chamber.
• To remove unwanted air bubbles in the chamber or gel add butanol in it.
• Insert a comb in between the space of the glass plate.
• Allow to set the gel. This polymerized gel is a “gel cassette.”
• Sample Preparation
  • Boil water and add 2-mercaptoethanol to the buffer sample.
  • Add the buffer solution with the protein sample to microcentrifuge tubes.
  • Take MW markers in separate tubes.
  • Boil the samples for a few minutes for complete protein denaturation.
• Electrophoresis
  • Remove the gel cassette and place it in the electrode assembly.
  • Fix the electrode assembly with the clamp stand.
  • To fill the gel wells, pour 1x electrophoresis buffer in the opening of the casting frame.
  • Add 20-30ml denatured sample in the well using a pipette.
  • Cover the tank with a lid.
  • Connect the unit and start the power supply.
  • Allow the sample to run for 1 hour at 30mA.
  • View the bands under UV light.

Applications of SDS-PAGE

Few of the applications of SDS-PAGE are stated below:

1. Western Blotting
2. Peptide mapping.
3. To measure the molecular weight of the molecules.
4. To estimate the protein size.
5. To analyze post-translational modifications.
6. To compare the polypeptide composition of different structures.
7. Protein ubiquitination.
8. To check the protein purity.
9. To analyze the size and number of polypeptide subunits.
10. To separate the HIV proteins in the HIV test.

Importance of SDS-PAGE

In molecular biology and biochemistry, sodium dodecyl sulphate polyacrylamide gel electrophoresis, or SDS-PAGE, is a crucial and often applied method.

1. Seperation of Protein: Proteins in a sample can be effectively separated using SDS-PAGE. It gives scientists a visual depiction of the protein composition and enables them to identify specific proteins within a complicated combination.
2. MW determination: SDS-PAGE allows the molecular weight of unknown proteins to be estimated by running known molecular weight standards alongside the sample.
3. Purity analysis: Protein samples may be tested for purity using SDS-PAGE. Impurities can be identified by contaminants or extra bands in the gel, which enables researchers to assess how well protein purification techniques worked.
4. Quantification of Proteins: Protein levels can be semi-quantitatively analysed since the intensity of the protein bands on the gel correlates with protein concentration.
5. Western Blotting: Western blotting is a technique that uses antibodies to identify and quantify certain proteins.

Conclusion- SDS-PAGE

SDS-PAGE or Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique used to separate proteins based on their molecular weight.

• In this method, protein molecules are separated based on their molecular weight.
• It is used in various branches of biology such as molecular biology, genetics, forensics, and biotechnology for protein separation.
• It separates protein molecules based on their electrophoretic mobility.

FAQs – SDS-PAGE

What is SDS-PAGE?

SDS-PAGE is an electrophoretic technique used to separate protein molecules based on their molecular weight.

Why is SDS added to Polyacrylamide gel?

The SDS breaks the disulfide bonds and modifies the protein structure. These modified structures and increased negative charge help to maintain the charge-to-mass ratio in electrophoresis.

Which Stain is used in SDS-PAGE?

To stain peptide bonds in the sample the Coomassie blue stain is used in SDS-PAGE. It stains the peptide bond blue.

What is TEMED?

TEMED is also known as Tetramethylethylenediamine. It is an ethylenediamine derivative.It is commonly used for polyacrylamide gel preparation in combination with ammonium persulfate.

What is Polyacrylamide?

Polyacrylamide is a synthetic linear polymer which is composed of acrylamide or a mixture of acrylamide and acrylic acid. It is water-soluble and used in pulp and paper industries.

What is the Function of SDS in Protein Extraction?

SDS is an anionic detergent that disintegrates the covalent bonds between the proteins and confers the negative charge to the protein according to its mass.

Which Buffer is used in SDS-PAGE?

The buffer used in SDS-PAGE is the SDS running buffer that is made up of Tris-HCl, SDS, glycerol, beta mercaptoethanol (BME) and Bromophenol Blue.