protein concentration problem - (Nov/02/2008 )

Hello,

I would be gratefull if someone could help me with my problem. I have tried to extract protein from plant samples to do western blotting but the protein concentration I will get from extractions is too low to perform western blotting. I have tried different types of buffers and tried to ground the samples very fine in liquid nitrogen before mixing with buffer. I have tried to concentrate the proteins with acetone precipitation but I only will got 2x higher amounts of protein after precipitation. The protein amount (10 ug) is too low to detect my protein of interest with antibody from blots. Has anyone some suggestions of how to get more protein or suggest some other more effective protein precipitation protocol. I know my antibody is working as I will get nice results with recombinant protein of interest.

may try to concentrate the protein of interest using spin column.

what's the volume protein's suspended in? I'm not really familiar with plants, and I'm just a beginner, but one of the postdocs in our lab used a "centrifugal concentrator" to pellet the protein and resuspend in a smaller volume... of course that was with an Ecoli recombinant