SDS PAGE or Sodium Dodecyl Sulfate-Polyacrylamide Gel electrophoresis is a method used to separate proteins based on molecular weight.
This method is widely used in forensics, genetics, biotechnology and molecular biology to separate protein molecules based on their electrostatic mobility.
Principle of SDS-PAGE
The SDS-PAGE technique states that a charged molecule placed in an electric field migrates to an electrode with another label. The separation of charged molecules depends on the relative mobility of the charged species.
Small molecules migrate easily due to low resistance during electrolysis. The structure and charge of the protein affect the speed of migration.
Sodium dodecyl sulfate and polyacrylamide eliminate the conformational and charge effects of the protein, and protein dissociation depends on the polypeptide chain length.
SDS is the eluent in the SDS-PAGE sample buffer. In addition to some reducing agents, SDS serves to break the disulfide bonds of proteins, which disrupt the tertiary structure of proteins.
- Electricity: It is used to convert AC current to DC current.
- Gels: These are laboratory made or commercially available pre-made gels.
- Electrophoretic chambers: Accessible chambers for SDS-PAGE gels should be used.
- Protein samples: Proteins are diluted using SDS-PAGE sample buffer and incubated for 10 minutes. A reducing agent such as dithiothreitol or 2-mercaptoethanol is also added to reduce disulfide bonds to prevent any tertiary protein stretching.
- Running buffer: Protein samples loaded on the gel are run in SDS-PAGE running buffer.
- Staining and Blocking Buffer: The gel is stained with Coomassie Stain Solution. The gel is then separated by a solution. Protein spots then appear under the eyes.
- Protein ladders: A reference protein ladder is used to identify proteins of interest based on molecular mass.
Protocol of SDS-PAGE
- To prepare the gel, all the ingredients except TEMED are combined.
- When the gel is ready to pour, add TEMED.
- The separation is poured into the gel mold chamber.
- Butanol may be added prior to polymerization to remove unwanted air bubbles present.
- The bracelet is inserted into the slots between the glass panel.
- Polymer gels are known as “gel cassettes”.
Applications of SDS-PAGE
- It is used to measure the molecular weight of a molecule.
- It is used to estimate protein concentration.
- used in peptide mapping
- It is used to compare the structure of polypeptides of different structures.
- It is used to assess protein purity.
- It is used for western blotting and protein ubiquitination.
- It is used in HIV testing to isolate HIV cells.
- Size and number analysis of polypeptide subunits.
- Analysis of post-translational changes.
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