Protein purification before mass-spec - need sample purification methods (Nov/15/2006 )
Hi, 
I have already purified a complex misture of proteins (cell lysates) into different fractions (by electro-elution) and I need to identify the protein present in one of these fractions.
The problem is that the fraction that I want is not purified enough and I think it is not possible to do mass-spec of a misture of proteins. I have aprox 1mg of total protein in this fraction.
What methods of purification can I use to purify my protein further?
Does somebody have any experience in size exclusion cromatography? Is this a good technic? How much can I recover from my protein sample?
Thaks in advance...
how did you fractionate to this point?
sec will not suit your needs if you already separated by size.
what is the condition of your protein. you may be able to separate by ion exchange or reverse phase.
in fact, if your ms is an lc-ms then you should be able to separate and determine your protein on the machine.
I have already purified a complex misture of proteins (cell lysates) into different fractions (by electro-elution) and I need to identify the protein present in one of these fractions.
The problem is that the fraction that I want is not purified enough and I think it is not possible to do mass-spec of a misture of proteins. I have aprox 1mg of total protein in this fraction.
What methods of purification can I use to purify my protein further?
Does somebody have any experience in size exclusion cromatography? Is this a good technic? How much can I recover from my protein sample?
Thaks in advance...
I would think of 2D gelectrophoresis; you seem to have enough protein to separate; at least, you need ca. 1 pmol of non-contaminated protein for Ms/Ms;
Thank you mdfemko & kosmodrom!
I already separated my protein by size in an SDS-PAGE. I dont know if my protein is suitable for ion exchage or reverse phase because i realy dont know witch protein I will detect.
The ms that we have here is a MALDI-tof MS, I dont think it has liquid cromatography attached. 
Sorry, but I am not very experienced in these matters. I will try to speek with the technician.
I belive that the solution that contains my protein is still very complex. This is why I want to purify my protein further, before using the ms... If SEC is not adequate, maybe I will try ion exchage... Can someone tell me were I can get this column ?
ge healthcare has the resource q column for anion exchange and resource s for cation exchange.