SDS-PAGE
SDS PAGE or Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis is a technique
used for the separation of proteins based on their molecular weight. It is a technique widely used
in forensics, genetics, biotechnology and molecular biology to separate the protein molecules
based on their electrophoretic mobility.
Table of Contents
Principle of SDS-PAGE
Materials Required
Protocol of SDS-PAGE
Applications of SDS-PAGE
Frequently Asked Questions
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 of the charged molecules depends
upon the relative mobility of charged species.
The smaller molecules migrate faster due to less resistance during electrophoresis. The structure
and the charge of the proteins also influence the rate of migration. Sodium dodecyl sulphate and
polyacrylamide eliminate the influence of structure and charge of the proteins, and the proteins
are separated based on the length of the polypeptide chain.
Role of SDS in SDS-PAGE
SDS is a detergent present in the SDS-PAGE sample buffer. SDS along with some reducing
agents function to break the disulphide bonds of proteins disrupting the tertiary structure of
proteins.
Materials Required
Power Supplies: It is used to convert the AC current to DC current.
Gels: These are either prepared in the laboratory or precast gels are purchased from the
market.
Electrophoresis Chambers: The chambers that can fit the SDS-PAGE gels should be
used.
Protein Samples: The protein is diluted using SDS-PAGE sample buffer and boiled for
10 minutes. A reducing agent such as dithiothreitol or 2-mercaptoethanol is also added to
reduce the disulfide linkages to prevent any tertiary protein folding.
Running Buffer: The protein samples loaded on the gel are run in SDS-PAGE running
buffer.
Staining and Destaining Buffer: The gel is stained with Coomassie Stain Solution. The
gel is then destained with the destaining solution. Protein bands are then visible under
naked eyes.
Protein Ladder: A reference protein ladder is used to determine the location of 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 poured, add TEMED.
The separating gel is poured in the casting chamber.
Add butanol before polymerization to remove the unwanted air bubbles present.
The comb is inserted in the spaces between the glass plate.
The polymerized gel is known as the “gel cassette”.
Also read: Applications of Biotechnology
Sample Preparation
Boil some water in a beaker.
Add 2-mercaptoethanol to the sample buffer.
Place the buffer solution in microcentrifuge tubes and add protein sample to it.
Take MW markers in separate tubes.
Boil the samples for less than 5 minutes to completely denature the proteins.
Electrophoresis
The gel cassette is removed from the casting stand and placed in the electrode assembly.
The electrode assembly is fixed in the clamp stand.
1x electrophoresis buffer is poured in the opening of the casting frame to fill the wells of
the gel.
Pipette 30ml of the denatured sample in the well.
The tank is then covered with a lid and the unit is connected to a power supply.
The sample is allowed to run at 30mA for about 1 hour.
The bands are then seen under UV light.
Applications of SDS-PAGE
The applications of SDS-PAGE are as follows:
1. It is used to measure the molecular weight of the molecules.
2. It is used to estimate the size of the protein.
3. Used in peptide mapping
4. It is used to compare the polypeptide composition of different structures.
5. It is used to estimate the purity of the proteins.
6. It is used in Western Blotting and protein ubiquitination.
7. It is used in HIV test to separate the HIV proteins.
8. Analyzing the size and number of polypeptide subunits.
9. To analyze post-translational modifications.
