Paul et al.:
A simple way to remove the Coomassie would be to use HILIC conditions
with a spin column (PMASCHY1203 at $3.50 ea.). Dilute the sample
with 3 parts MeCN and bind it to the column. The Coomassie will
elute in the loading solvent. Then recover the peptides with a step
to 5% MeCN with 50 mM ammonium acetate. This will assure that even
phosphopeptides would be eluted. For more information look at the
HILIC pages at http://world.std.com/pdf/pdf.html or Jen– et al. Anal.
Biochem. 215 (1993) 292 (Removal of SDS and salts from electroeluted
proteins).
Best regards,
Amos Heckendorf
>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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