Hello guys, hope you all are doing great. Also I assume that the last blog on exam tips might have helped you with exam preparation.
Moving ahead.. the UG’s have already started with their exams. Now it’s time for the PG’s to show their hardwork and efficiency. So for today the topic that we would cover is SDS-PAGE.
- Electrophoresis in acrylamide gel is frequently referred to as PAGE (POLYACRYLAMIDE GEL ELECTROPHORESIS).
- The components used in PAGE
- PAGE combined with SDS is the most widely used method for analysing protein mixtures quantitatively.
- SDS-PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis) is a discontinuous system.
- In SDS-PAGE, the gel consists of two parts namely, Resolving gel (3/4th) & Stacking gel (1/4th).
- The separating gel or resolving gel is first poured into the electrophoretic apparatus and allowed to set .Then the stacking gel is poured on the top of the separating gel. Wells for loading the sample are made with the help of comb.
- Samples of proteins of known and unknown molecular weight are loaded in the wells.
- The stacking gel allows the protein to move freely and concentrate over the separating gel under the influence of the electric field. Proteins continue their movement towards the anode.
- Since proteins have the same charge per unit length, all proteins travel with the same mobility. However, as they pass through the separating gel, the proteins separate owing to the molecular sieving properties of the gel.
- The smaller proteins move fast as they can pass through the pores of gel. But the larger proteins move slowly since they are retarded by frictional resistance due to sieving effects of gel.
- When the dye reaches the bottom of the gel, the current is turned off. The gel is removed and stained with staining solution like COMASSIE BRILLIANT BLUE.
- A plot of the distance migrated versus log of the molecular weight gives a straight line. Hence, if a protein of unknown molecular weight is electrophoresed with two or more proteins of known molecular weight, the molecular weight of unknown protein can be calculated to an accuracy ranging between 90 & 95 percent. This is the most common way of estimating the molecular weight of protein subunits.
FOR FURTHER UNDERSTANDING OF THE ABOVE TOPIC VISIT THE LINK BELOW,
REFRENCE: Bioinstrumentation by M.H.Fulekar