different western results when using acetone prep vs crude protein - (Feb/18/2008 )
I am working on a nuclear protein of plant. I made a polyclonal antibody against 2/3 of my protein. However, in western blot experiments, my protein can be only detected from the acetone precipitated protein. If I load crude protein extracted by Tris-Urea buffer, the band is gone on western. I used the same material, the same SDS-PAGE and Western protocol, and the only difference was method of protein extraction.
why is the difference between acetone precipitation protein vs crude protein extracted in Tris-Urea buffer? What is the possible explanation of my strange results?
a couple of possibilities:
the urea denatures your protein when it is extracted as opposed to your crude lysate. this is a less likely problem because you denature the protein when you apply it to the gel.
maybe more likely, urea breaks down and carbamylates the protein.
I think it could be possible that there is something in the crude extract that is interfering with ab that is removed during the acetone precipitation. Do you use the tris-urea buffer to extract the sample and then do the acetone precipitation? Maybe you could try a different lysis buffer that is strong to break open the nucleus.
Thanks for all your reply. I will try new buffer.