Question

1. How does 2D gel electrophoresis analysis separate protein?

Professor gave some hints: Technique of 2D gel electrophoresis & What separates proteins by individual molecules by approaching it in two different ways.

Answer 1

2D electrophoresis, used to separate the protein . Here mixture of protein is separated in two directions by using two property ( one is charge and second is mass of protein).

2-D electrophoresis proteins are separated in two dimensions. It begins with electrophoresis in the first dimension folloed by separtion of molecules perpendicularly from the first thus created an electropherogram in the second dimension.

first dimension, - molecules are separated based on their isoelectric point. A  gradient of pH is applied to a gel and an electric potential is applied across the gel. It makes one end of gel more positive than the other end . Except the isoelectric pH , protein will be charged at all pH values . Now they will move according to their charge, f they are positively charged, move to negative end of the gel and if they are negatively charged , move to positive end of the gel. The proteins will move along the gel and will accumulate at their isoelectric point; that is, the point at which the overall charge on the protein is 0. Thus leads to sepration of protein.

Isecond dimension - molecules are separated at 90 degrees from the first electropherogram according to molecular mass using SDS gel. Treatment of SDS leads to denature the protein and provide overall negative charge to the protein. Now protein will be separated on basis of mass.

Probability of any two molecules will be similar in two distinct properties, will be negligible. Thus molecules are more effectively separated using  2-D electrophoresis than in 1-D electrophoresis.

Rough image has been attached for better understanding