Linear vs conformational eptiopes on Western Blot

[thinkingoutloud]

Short version of my question:

Does anyone know if proteins can gain some secondary or tertiary structure following or during transfer to PVDF membrane after being run using SDS-PAGE


I suspect this is the case as I have 5 monoclonal antibodies that all blot. I belive some of these monoclonal antibodies are binding to conformational epitopes and yet I am told that in an SDS-Page and Western Blot the protein is in a linearized form. Does anyone know anything about this?

[danfive]

Under Laemmli conditions your proteins are linearized as you say. So your Antibodies are binding to denatured protein. If you care to know if they will also bind to biologically active protein, perform native-PAGE and the same WB.

[labgnome]

How certain are you that the epitopes are conformational? Also, I have performed western blotting using a monoclonal antibody which recognises only a conformational epitope, and on a denatured protein, there was a faint signal, but there neverthless. I think that, depending on the amount of protein you use, some may retain the conformation neccessary for recognition. Even i fthe majority of protein is denatured you may still see a faint signal

[danfive]

Originally Posted by labgnome

I think that, depending on the amount of protein you use, some may retain the conformation neccessary for recognition. Even i fthe majority of protein is denatured you may still see a faint signal

I totally agree, you have to perform the technique correctly to avoid this problem.

[mj_carp]

If you want tot be sofisticated you can also perform a 2D gel and blot it with your antibody. Then you will get not only size but potetial modifications. Also, form a 2D gel you can cut out the spot ( when you do a duplicate 2D gel, you stain one and blot the other and usperimpose them) and do Mass spec to identify peptides form your protien- confirming to a certain poitn the identity of your proteins. Good luck.