Paul,
I have personally not used the method describe by Jane for removal of Coomassie
Blue stain from an SDS PAGE band. The protocol that was outlined uses a high
percentage methanol and for what appears to be prolonged and repeated exposures.
I do not doubt that this will in fact destain the gel or band (the
methanol/acetic acid concentration is very similar to that of many destain
recipes) however, I would question the recovery of protein from the SDS PAGE
band following this procedure. You may wish to consider the following:
What is the estimated amount of protein present in the band of interest? This
can be estimated by the intensity of staining or better yet if the amount of
protein loaded to the SDS PAGE lane is known an estimate can be made for the
band of interest based on purity observed.
How much protein is required for downstream analysis? What sort of recovery can
you live with?
Your original posting stated that you had already extracted the protein from the
SDS PAGE band and are interested in removing Coomassie blue dye from the extract
(removing Coomassie Blue that is bound to the protein). Unfortunately, I can
not offer a solution to you. I would however like to comment about the use of a
Zip tip or C18 resin. I have completed numerous in-gel digestions of proteins
that are stained intensely with Coomassie Blue and collected purified peptides
from C18 RP-HPLC. Coomassie Blue does bind to C18 and elutes very late (very
broad) with a high percentage of ACN. If the staining was completed using
Coomassie Blue R as opposed to more purified Coo-Blue G, than you should expect
to see more Coo Blue related peaks.
I do not agree with the notion that your extracted protein will not bind to the
C18 resin. It may bind and elute at a lower Acetonitrile concentration, or it
could bind strongly and you may suffer large losses.
Having said all that, I can offer one possible solution to your problem if the
investigator is willing and able to work with you toward solving this problem.
Repeat the SDS PAGE separation, use Zinc Reverse staining to detect the band of
interest, destain in Tris buffer or 2% Citric acid, briefly wash the destained
band in water, and perform a passive elution from the washed band. The staining
and destaining is rapid and extremely simple (a kit is available from Bio-Rad if
you do not wish to prepare your own reagents). No harsh reagents are used that
fix the protein of interest in the SDS PAGE gel, and no dyes are bound to the
protein to interfere with downstream analysis.
Other issues to consider: Normally recovery of protein using passive elution is
high, but generally the eluted protein is captured onto PVDF membrane. In the
case where no PVDF membrane is being used, it will be neccessary to minimize
volumes of elution buffers used and decrease exposure time and surface areas to
plastics. Again, the question of how much protein can you afford to lose for
your downstream analysis is important.
The problem you posted is really quite interesting for a couple of reasons: It
demonstrates the importance of good communication between the investigator and
the analyst at the beginning of an experiment; Coomassie Blue staining is, in
this case, clearly not the best way to proceed given the type of downstream
analysis required. Given the intensity of the staining that you described, even
a modified Coomassie Blue staining procedure with limited exposure to the dye
and fixatives may have been more suitable. This problem also demonstrates the
importance of continued discussions with the investagator aimed at the
successful resolution of a problem. "Help me...Help you".
Good Luck,
Raymond Boynton
N-terminal Protein Sequencing Facility
Biogen, Inc.]
Cambridge MA, 02142
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