Question

Why do we use SDS? Why PA? why GE? Why Lammeli buffer? Why running buffer?

Answer 1

Sample, that is, protein is loaded onto the electophoteic gel, during analysis, in the form of buffer called sample buffer. SDS is sodium dodecyl sulphate, a substance used in sample buffer during polyacrylamide gel electrophoresis (PAGE). It is an anionic detergent. It denatures secondary and non–disulfide–linked tertiary structures of proteins and subunits and make them negatively charged so that each will separate based on its size.

Polyacrylamide (PA) gel electrophoresis (GE) is an analytical tool to separate components of a protein mixture by molecular sieving effect based on its size. The technique utilizes the ability of charged protein in a gel medium to migrate towards the oppositely charged electrode (anode) in an electric field. After the visualization by a staining (protein-specific) technique (usually with bromophenol blue), the size of a protein can be calculated by comparing its relative migration distance (Rf) with that of a known molecular weight ladder(marker). From the obtained value, logarithm of the molecular weight of the protein can be plotted against the Rf graphically. Interpolating the value from this graph will then give you the molecular weight of the unknown protein band.

Polyacrylamide is a polymer (-CH₂CHCONH₂-) formed from acrylamide subunits. It can be synthesized as a simple linear-chain structure or cross-linked, typically using N,N'-methylenebisacrylamide. It is highly water-absorbent, forming a soft gel when hydrated, used in such applications as polyacrylamide gel electrophoresis,

Laemmli sample buffer (by Laemmli in 1970) (prepared by is used in the Laemmli SDS-PAGE analysis for protein sample preparation. A protein sample is mixed with the 2Xsample buffer (1:1) and heated in boiling water for 2-5 min. The 2-mercaptoethanol is to be added to reduce the intra and inter-molecular disulfide bonds. This buffer ensures optimal band resolution when preparing protein for SDS-PAGE with Tris-glycine-SDS running buffer.

Running Buffer in gel electrophoresis is used to provide ions that carry a current (by electrolyte ions migrating towards electrodes) and to maintain the pH at a relatively constant value. Usually Tris-glycine buffer system is used in SDS-PAGE gel electrophoresis.