In article <9510311542.AA22652 at umailsrv1.UMD.EDU>, Lynne_A_WHITEHEAD at UMAIL.UMD.EDU (lw75) says:
>>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!
>U. of Maryland - Microbiology Dept.
cut out the band from the gel, mince the gel to as small pieces as you can
get, and inject it subcutaneously into the rabbit. Most probably you will
obtain a good antiserum.