elution of monospecific antibody

Dima Klenchin klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Fri Nov 17 16:52:34 EST 2000

In article <3A08053B.BF62C4FB at uni-tuebingen.de>, Tressa Jacob <tressaj at uni-tuebingen.de> wrote:
:I am working with 6X his tagged DHFR consrtuct  for my peptide. I had
:managed to purify my protein. In order to raise antibodies I  run 13%
:SDS PAGE and cut the right band out and send it to the company to raise
:I obtained my antisera and I checked it to see whether it recognises my
:antigen....it does recognise upto 150ng purified protein but not10ng.

What dilution did you use?

:I thought of making it monospecific, I did so using the normal procedure
:; used 70 ╣g of proteins,tranfered  overnight and then cut the band of
:interest from the filter  and proceeded with blocking in 5% blocking
:solution in TBS-TritonX, then washing it and then incubating it with my
:antibody (1:1)2.5% blocking solution in TBS-T for 2 hours at RT and then
:washing 4X with TBS-T, then eluting it in 100mM Glycine for 10min and
:then immediately neuterlising it in 1M Tris pH 8.0.
:BUT::::::: I do not get any protein when I check it with Bradford
:my question is::::::
:how would I know if my antibody is still bound to the membrane??? 

You can probe with anti-rabbit secondary to find out.

:or is
:it washed off???? or it has not bound to the protein at all.
:Do you think I should throw off the antisera as the affinity is too

I would say YES. Coomassie can see 10-20 ng of protein, so
if your antisera (at reasonable dilution) can detect only 150 ng, 
it is basically not worth it. 

Crap happens - the rabbit could have been suffering from 
immunodeficiency, the company could have screwed up somewhere, 
your antigen might be very poorly immunogenic, but...

I think most likely you did not have enough of the antigen to really
hyperimmunize the rabbit and get nice antiserum. You have recombinant
expression in E.coli - what's the problem then? Why can't you 
_really_ purify it on a larger scale and provide 1-2 mg of it in very 
pure form? Then, barring poor service, in all likelyhood you will end
up with a very good serum by second/third bleed. 

        - Dima

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