Recovering proteins from Western Blots

Virginia Dress Dress at biosci.arizona.edu
Thu May 5 19:07:27 EST 1994


In article <Berry.58.2DC8BE89 at biochem.unp.ac.za>, Berry at biochem.unp.ac.za
(Ronald Berry) wrote:
> 
> Hello All
> 
> I have a Western blot of a protein, which has been immunodetected and 
> stained with ABTS. I want to recover the original protein intact in order to 
> do an electrospray mass spec study on it. Therefore, I need to firstly strip 
> off the antibody (and precipitated ABTS stain?), and then release the 
> protein.
> 
(deletion)
> Needless to say, I have *VERY* little sample available, and so trial and 
> error shots are out of the question. 
> 
> All contributions will be gratefully received.
> 
> Cheers,
> Ron Berry

Getting protein back from the nitrocellulose sounds pretty unlikely to me,
but
it seems that MAYBE you could electroelute it from the nitrocellulose, use
some
SDS to help the process.  I'm saying this based on the idea that you can
transfer on and then 'through/off' the nitrocellulose if you transfer too
long.
But like the other post-er said, you did block the blot with some
non-specific
protein (probably) and will now have that contaminant.  I have heard of
people
"blocking" with Tween-20 or Triton X-100, but when I tried Tween it
definately
did not work.  I don't know what conditions you need to do the mass spec,
ie.
would SDS interfere?  How much sample do you need for the mass spec?  Do
you
really have enough on your blot to do mass spec with?  What sort of yield
would
you need?  At best you could hope for maybe 50% recovery, but will you need
more
like 95% recovery?

Sounds like a really tough problem.  The Hoefer 6-pac eluter set-up would
be
nice because you elute into microfuge tubes and you could keep your volume 
small.  You could test my idea with some protein of similar MW to check the
efficiency, etc.   If you try this let me know if it works.

Good luck,

Virginia Dress
Dress at biosci.arizona.edu



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