How big should a tissue sample be for western blotting?

Answer 1

Depends on the the type of tissue, extraction/purification method, and the distribution of your protein of interest. It also helps if you have a good antibody, and you are using a secondary detection system that amplifies your signal (i.e. rabbit Anti-"protein" IgG and goat Anti-rabbit IgG FITC or HRP conjugate) then you would need very little tissue. This is because most of the time your secondary antibody cocktail is polyclonal, and thus will react with many different parts of your protein rather that one specific small piece like a monoclonal antibody. If your tissue has an abundance of your protein of interest you need even less tissue. Basically were talking about tens of thousands of cells, or in more obvious terms very low milligrams to micrograms. Also a big factor is how well you can purify and concentrate your protein from your tissue. If this is for a homework packet or a lab tell your instructor to be specific, if you are looking for actual help with an experiment then let me know, I am a graduate student in biology and for my thesis research do lots of westerns and have many protocols and tricks that can be helpful. Cheers.

Answer 2

The above answer with regards to the help of proteolysis of the protein of interest is maximum acurate, even nevertheless maximum lysys buffers arenothing better than isotonic detergent mxtures used o solubalise cellular membranes. This technique can bring about the activation of proteases and particularly some lysis buffers can comprise protease inhibitors to help shrink digestion. Denaturation of the protein won't be a topic till working a close-by gel, because of the fact the pattern might have had to have been heated with SDS and so on previously being loaded into the wells of the gel.

Answer 3

Mine was in 10ml at 1:2000 dilution.