elution of monospecific antibody

Tressa Jacob tressaj at uni-tuebingen.de
Tue Nov 7 08:35:55 EST 2000


hello!
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
antibodies.....
I obtained my antisera and I checked it to see whether it recognises my
antigen....it does recognise upto 150ng purified protein but not10ng.
I thought of making it monospecific, I did so using the normal procedure
; used 70 µg 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
method.

my question is::::::

how would I know if my antibody is still bound to the membrane??? 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
low??????

kindly help.


Tressa.







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