Paul,
The best way to get rid of the coomassie is _before_ the extraction procedure. If you still have some of the intact band, wash it sequentially as follows:
1. 100mM ammonium bicarb
2. 50% MeOH + 10% HOAc
3. 40% EtOH
Repeat until the band is clear and then do your extraction procedure. I find that it takes 5 to 6 cycles through these steps to get rid of the coo blue completely.
If you have none of the original band then you could try zip tips to pull out the coomassie. I find that the C18 tips will collect the dye nicely and they shouldn't bind your protein. Try pullling out the dye with the C18, and then zip tip the remaining sample with a C4 tip. Just a suggestion, I haven't actually tried this!
Good Luck
-Jane
Jane A. Nagel, MSc
Genetics Institute
87 Cambridge Park Dr
Cambridge, MA 02140
617 665 8597
fax 617 665 8878
1 888 577 1500 x8597
>>> pds <pds@fossil.biochem.ualberta.ca> 12/08 9:43 am >>>
Recently someone has brought me a gel band which is stained very dark with
coomassie.They want the mass of the intact protein.I extracted the protein
according to the paper of Cohen and Chait (Ana. Biochem.
1997,247,257-267)using formic acid:water:isopropanol(1:3:2) but get a very
weak and broad signal.I suspect that it is the high concentration of coomassie
that is interfering.Does anybody know of a procedure that will reduce the
amount of coomassie in the sample.Thanks.
sincerly,
Paul Semchuk
Institute for Biomolecular Design
University of Alberta
Edmonton,Alberta,Canada
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