protein extraction from SDS-Page

lw75 Lynne_A_WHITEHEAD at UMAIL.UMD.EDU
Tue Oct 31 10:44:09 EST 1995


Hi there!

I need help!  I have a ton of an agarase protein that I an trying to purify
in my 8% SDS-PAGE, however I cannot extract it from the gel.  We think that
the protein is aggregating or precipitating in the gel. The protein will be
used to raise antibodies against it.  I'd like to retain activity if possible
(the lysis buffer doesn't contain mercaptoethanol, the sample is not heated,
and the SDS can be removed), but at this point I'd settle for just getting it
out of the gel even if it's denatured. Any suggestions are welcome!
I have already tried:
1. eluting the protein from the band overnight into buffer at room temp after
crushing the gel.

2.  electroeluting the protein with very poor yield.

3.  transferring the protein to nitrocellulose and dissolving the
nitrocellulose with acetone as described in "Antibodies".  After the acetone
evaporated away and I tried to resuspend the protein in buffer, but the
nitrocellulose precipitated on the walls of my glass tube.  I know that
there is a ton of protein there because I india ink stained a portion of the
nitrocellulose and got a huge band.  Does anyone recommend another
proceedure for this?

Thanks for your help!

Lynne Whitehead
U. of Maryland - Microbiology Dept.




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