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.
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.
Table of Content
- Define SDS-PAGE
- What is Electrophoresis?
- What is SDS-PAGE?
- Principle of SDS-PAGE
- Materials Required
- Procedure of SDS-PAGE
- Applications of SDS-PAGE
- Importance of SDS-PAGE