getting rid of protein above 60 kDa - (Jan/07/2008 )
Hello,
Do you know any method to get rid of proteins above 60 kDa in protein extract? I want to do SDS page and cut the band, but as my samples come from mammalian cell lysates, there is a lot of additional stuff I don't need. It would be easier to see what you cut on the gel. My protein of iterest is about 50 kDa.
Many thanks for those who will save my life by answering this question !!! 
I would try to purify a little bit with anion exchange and gel filtration.
Depends on the amount of sample you have
There are centrifuge ultrafiltration filters at 50KDa and 100 KDa you can use to process small samples. Pull, millipore makes these and you can get from distributers like VWR or Fisher Sci.
Do you know any method to get rid of proteins above 60 kDa in protein extract? I want to do SDS page and cut the band, but as my samples come from mammalian cell lysates, there is a lot of additional stuff I don't need. It would be easier to see what you cut on the gel. My protein of iterest is about 50 kDa.
Many thanks for those who will save my life by answering this question !!!
filtration with cut-off of 50 kDa will enrich proteins of >60 kDa;
but I think you do not need this if you separate your proteins in 2D gelectrophoresis; most proteins run in single spots or strains which is due to cut out distinctly (f.i. for sequencing)
gel filtration (eg- sephacryl s-200) would be best for your purpose.
fractionate, run fractions on a gel, then pool those fractions that meet your requirements and concentrate (if necessary).
thank you all! I have something to try now!
Unfortunately, I can't do 2D (no equipment in the lab or nearby).
Do you know any biochemical methods? Some methanol:acetic acid or other mixtures ?
do you mean "chemical" methods like organic extraction or phase partitioning?
not for protein (you may be able to crystallize from acetone).
Unfortunately, I can't do 2D (no equipment in the lab or nearby).
Do you know any biochemical methods? Some methanol:acetic acid or other mixtures
?you may lose a lot of time in enriching or purifying a distinct protein...better go straight ahead; there are numerous commercial services who will do 2D-GE if you prepare the probe; or you will find collaborators for a paper; your university/medical school etc may have a proteomics facility...