protein extraction from SDS-Page
fgarbrec at HENDRIX.JCI.TJU.EDU
Tue Oct 31 11:25:42 EST 1995
When you use the nitrocellulose method, your protein is probably
irreversibly bound to the nitrocellulose. You should make a slurry
of nitrocellulose powder (with your protein attached) and just inject
that into the animal you are immunizing. For what it is worth, I
don't know why your electroelution should not work if you are able to
blot your protein out onto NC. You could also just crush your
polyacrylamide gel band (after cutting it out), make a slurry of that
and use it for injections.
> To: methods-and-reagents at net.bio.net
> From: Lynne_A_WHITEHEAD at umail.umd.edu (lw75)
> Subject: protein extraction from SDS-Page
> Date: 31 Oct 1995 07:44:09 -0800
> 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.
Thomas Jefferson University
fgarbrec at lac.jci.tju.edu
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