Download
1 / 6
E N D
SDS- PAGE M.Prasad Naidu MSc Medical Biochemistry, Ph.D,.
Separation of Proteins & purification of proteins According to size & mol.wt SDS is anionic detergent CH3-(CH2)11-O-SO3- - Na+ Sodium Dodesyl Sulphate Polyacrylamidegel Electrophoresis
Samples to be run on SDS-PAGE are first boiled with 0.1M β-mercaptoethanol + 1% SDS for 5 min β-mercaptoethanol reduces –S-S- bonds and causes denaturation SDS binds strongly on proteins (Aveg: 1SDS + 1 AA) 1.4gm SDS/gm protein Protein is denatured and gets –ve charge due to binding with SDS This makes the internal charges negligible and the entire protein gets –ve charge Procedure
Sample is boiled in a sample buffer Sample buffer contains Tracking dye – Bromophenol blue BB allows the passing of proteins through the gel All the proteins are –vely charged anode Mobility α mol.wt Sieving effect of the gel causes movement of smaller size particles easily and vice versa Proteins can be visualized by treating the gel with Coomassive brilliant blue CBB binds to protein but not the gel Each band on gel represents a protein Smaller proteins are found near the bottom of gel and larger proteins at the top Procedure
PAGels have very good mol sieving effect than starch gels Adsorption of proteins is negligible Separation of proteins, & Nucleic acids A combination of Agarose & Acrylamide gel is used for the separation of High mol wt DNA Determines mol wt of proteins Purity of mixture of isozymes Applications
