Antibody Problems

patingsadagat via methods%40net.bio.net (by arnigambe from gmail.com)
Tue Jan 8 05:22:10 EST 2008


Dear All

I've been trying to produce antibodies against my protein of interest.
At first, I produced a His-6x tagged protein in E. coli and purified
the resulting protein using several purification methods.
We then sent the protein samples to a local antibody production
company. After 2 months, they sent us sera from rabbits before
immunization (0 W), 4, 6, 7 and 8 weeks after immunization. They also
sent a protein A/G-purified fraction of the 8W serum.
However, when I used the serum for immunostaining, I obtained
unexpected localization patterns which was similar to that of alpha
tubulin localization in HeLa cells. I therefore tested all fractions
using tubulin protein and to my dismay, all, including the 0W sera
could detect the tubulin protein.
I do not totally comprehend just what is going on. Should I continue
my attempts to purify my antibody of interest using the recombinant
protein (of interest), or is this a futile exercise?

Thank you in advance.


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