SDS Page

Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis

SDS PAGE also known as Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique used for separating the proteins based on their molecular weight. It is a widely used technique in forensics, genetics, biotechnology, and molecular biology to separate the protein molecules based on their electrophoretic mobility.

Principle of SDS-PAGE

The principle of SDS-PAGE states that a charged molecule migrates to the electrode with the opposite sign when placed in an electric field. The separation will take place as the mobility of the charged species. The tiny molecules tend to move faster due to their less resistance at the time of electrophoresis. The rate of migration influences the structure and the charge of the protein. 

Sodium dodecyl sulphate and polyacrylamide help to eradicate the influence of structure and charge of the proteins, and the proteins are separated based on the length of the polypeptide chain.

Function of SDS in SDS-PAGE

SDS can be defined as a detergent present in the SDS-PAGE sample buffer. The function of SDS is to break the disulphide bonds of proteins disrupting the tertiary structure of proteins along with some reducing agents. 

Materials Required

  • Power Supplies: It is used to convert the AC current to DC current.

  • Gels: These are either self prepared in the laboratory or are purchased from the market.

  • Electrophoresis Chambers: The chambers of the SDS-PAGE gels that fit well should be used.

  • Protein Samples: The protein is dissolved with SDS-PAGE sample buffer and boiled for 10 minutes. A reducing agent such as dithiothreitol or 2-mercaptoethanol is also added to minimize the disulfide linkages to prevent any tertiary protein folding.

  • Running Buffer: The protein samples filled on the gel are run in SDS-PAGE running buffer.

  • Staining and Destaining Buffer: Coomassie Stain Solution is used to stain. A destaining solution is used to destain a gel. Protein bands can then be observed with naked eyes.

  • Protein Ladder: A reference protein ladder is used to locate the protein of interest based on the molecular size.

Protocol of SDS-PAGE

Preparation of the Gel

  • All the reagents are combined, except TEMED, for the preparation of gel.

  • When the gel is ready to be put, add TEMED.

  • The gel used to separate is poured in the casting chamber.

  • You need to put butanol before polymerization to remove the unwanted air bubbles present.

  • Between the glass plate, the comb is inserted. 

  • The “gel cassette” is the  polymerized gel. 

  • Sample Preparation

  • Boil some water in a beaker.

  • Add 2-mercaptoethanol to the sample buffer.

  • Put the buffer solution in microcentrifuge tubes and add protein samples to it.

  • Take MW markers in separate tubes.

  • Bring the samples to boil for less than 5 minutes to completely denature the proteins.

Electrophoresis

  • The gel cassette is put off from the casting stand and placed separately in the electrode assembly.

  • The clamp stand is fixed to the electrode assembly.

  • 1X electrophoresis buffer is added to the opening of the casting frame to fill the wells of the gel.

  • Fit 30ml of the denatured sample in the well.

  • The tank is covered with a lid and the unit is attached to a power supply.

  • The sample is made to run at 30mA for about 1 hour.

  • UV light are used to observe the light.

FAQ (Frequently Asked Questions)

1. What is SDS page?

SDS PAGE also known as Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique used for separating the proteins based on their molecular weight. It is a widely used technique in forensics, genetics, biotechnology and molecular biology to separate the protein molecules based on their electrophoretic mobility.

2. What are the materials used in SDS page?

  • Power Supplies: It is used to convert the AC current to DC current.

  • Gels: These are either self prepared in the laboratory or are purchased from the market.

  • Electrophoresis Chambers: The chambers of the SDS-PAGE gels that fit well should be used.

  • Protein Samples: The protein is dissolved with SDS-PAGE sample buffer and boiled for 10 minutes. A reducing agent such as dithiothreitol or 2-mercaptoethanol is also added to minimize the disulfide linkages to prevent any tertiary protein folding.

  • Running Buffer: The protein samples filled on the gel are run in SDS-PAGE running buffer.

  • Staining and Destaining Buffer: Coomassie Stain Solution is used to stain. A destaining solution is used to destain a gel. Protein bands can then be observed with naked eyes.

  • Protein Ladder: A reference protein ladder is used to locate the protein of interest based on the molecular size.

3. What are the applications of SDS page?

The applications of SDS-PAGE are as follows:

  • It is used to measure the molecular weight of the molecules.

  • It is used to measure the size of the protein.

  • Used in peptide mapping

  • It is used to compare the polypeptide composition of different structures.

  • It is used to eradicate the pureness of the proteins.

  • It is used to separate the HIV proteins in HIV test

  • Analyzing the size and number of polypeptide subunits.

  • To analyze post-translational modifications.

4. What is the function of SDS in SDS page?

SDS can be defined as a detergent present in the SDS-PAGE sample buffer. The function of SDS is to break the disulphide bonds of proteins disrupting the tertiary structure of proteins along with some reducing agents.