Protocol for SDS-PAGE Analysis
|Author: Arun Malik, Harnish, Priyanka Chopra, Stanzin Angm*|
|Affiliation: National Agri-Food Biotechnology Institute, Mohali, Punjab, India|
|Source: Protocol Online|
|Date Added: Thu Feb 28 2013|
|Date Modified: Sun Mar 10 2013|
|Abstract: This protocol described the validated protocol in our lab for SDS-PAGE analysis of proteins.|
PROTOCOL FOR SDS-PAGE ANALYSIS
Arun Malik, Harnish, Priyanka Chopra, Stanzin Angmo and Hariom Yadav[*]
National Agri-Food Biotechnology Institute, Mohali, Punjab, India
SDS-PAGE technique broadly used in molecular biology, used to separate proteins according to their length of a linear polypeptide chain. The general electrophoresis technique cannot use to measure molecular weight of biological molecules but this technique mostly used to measure the molecular mass. The term electrophoresis means movement of charged particles to an electric field. In an electric field movement of proteins towards electrode of opposite charge. The separation of proteins according to their shape, size takes place.
SDS-PAGE conducted on casting the gels to avoid trouble and hazard of working with acryl amide. In this protocol presence of SDS and beta-mercaptoethanol which is used to denature the proteins in to their subunits.
Figure. SDS-PAGE Process Flow
Stock solutions: resolving gel for 12% solution
25 mM Tris-HCL
200 mM Glycine
0.1% (w/v) SDS
Approximately volume of water keeping below 1 liter that depends on electrophoresis system.
10% (w/v) SDS
10 mM Dithiothreitol or betamercapto-ethanol
20% (v/v) Glycerol
0.2 M Tris-HCL, Ph-6.8
0.05% (w/v) Bromophenolblue
Gel pouring into miniprotein cassette
Protein analysis and detection
Visualize the protein using Coomassie Brilliant Blue, silver stain, or we can use any other protein stain.
[*] To whom correspondence should be made: firstname.lastname@example.org