Western Blot

[Bio2013]

Hi BIologists,
I have a question, how can we know about the size of protein in the western blot? I know the full lenght, but after WB, I am seeing band with a different size!
Thanks for your help.

[JackBean]

What about post-transcrioptional modifications?

[Bio2013]

What about post-transcrioptional modifications?

Actually, I do not know exactly how to measure post translational modifications and reduce them from the full size of the protein, can any body help me with that? would post translational modifications be same for all proteins, and how can I find out about that?
thanks

[JackBean]

Those affecting size on SDS-PAGE would be mostly glycosylation or proteolysis, depending on the size of your band. Or the mobility of your protein is affected for some reason. Could you measure size of your protein on MALDI MS?

[Bio2013]

Those affecting size on SDS-PAGE would be mostly glycosylation or proteolysis, depending on the size of your band. Or the mobility of your protein is affected for some reason. Could you measure size of your protein on MALDI MS?

sorry, but I do not know what MALSI MS is.

[JackBean]

mass spectrophotometer

[MarkHolland]

Separating Proteins by Size

To begin characterizing a protein, it is often useful to be able to separate it from as many other proteins in the mixture as possible. One of the first techniques that can be used is to separate it according to how big it is, otherwise known as its molecular weight. When extracts are prepared from a cell, detergents can be used to make the proteins unfold into the linear chain of amino acids. Using a detergent such as sodium dodecyl sulfate (SDS) makes proteins unfold and gives them a net negative charge that is directly related to their molecular size. Using this feature, Dr. U. K. Laemmli developed a system of gel electrophoresis through a plastic medium (polyacrylamide) that easily allowed for proteins to be separated by an electric current strictly according to their overall mass. (1)