Question

In the Western blot experiment, )Why are proteins treated with ionic detergent (SDS), reducing agents (DTT), and heat before SDS-PAGE Why do SDS-coated proteins migrate in an electric field? e molecular mass of myosin light chain I is approximately 22 kD, myosin heavy chain is 200 kD ane actin is 42 kD, Which proteins will migrate fastest through the gel? Why?

Answer 1

1) SDS loading buffer contains SDS and reducing agents such as beta-mercaptoethanol or DTT. SDS disrupts the tertiary structure of proteins and brings them to the linear shape molecules. SDS also imparts a negative charge to protein masking their intrinsic charge. Thus, now proteins are uniformly negative charged proportional to their molecular weight. Now the protein will migrate according to molecular weight on SDS gel. Heating just speeds up the process of denaturation process of process. SDS coated proteins are negatively charged, they move toward a positive charge generated by an electric field.

2) fastest will be 22kd myosin light chain 1 protein followed by 42 kd actin and 200kd myosin heavy chain protein. The explanation is the same as in question one