[Protein-analysis] Smear in Immunoprecipitated Westerns

novalidaddress at nurfuerspam.de novalidaddress at nurfuerspam.de
Thu Feb 16 07:10:45 EST 2006


Dear Experts,

We detect protein phosphorylation by western blotting. To confirm the
identity of the detected bands, we are runnung immunodepletion
experiments (IP to remove protein from the lysate, expect none of the
phosphorylated protein in the supernatant but all in the precipitate).

In cell culture experiments, especially with transfected cells, it
works well, probably due to the abundance of the desired protein.

However, when working with biopsy samples (skeletal muscle) we get lots
of smear on the film, making their evaluation basically impossible.
This probably due to less amount of desired protein present in the
samples. So the smear amight always be present, but only becomes
visible when the ECL is exposed for a time long enough.

By doing control experiments omitting different parts of the reagents,
we conclude that the problem arises from the interaction of agarose
with the antibody. We also tried differerent protein G agaroses and
protein a/g agarose

We tried different antibodies for the IP, also against different
proteins, incubated the beads with Laemmli Buffer (actually, it's not
real Laemmli, we replaced Tris by phosphate) at 37degC for 30 mins and
removing the agarose beads before boiling the sample, did boiling the
samples with agarose at different temperatures (99, 85, 70 degC) and
got the more smear the lower the temp was.

The buffers for washing the agarose contains BSA (might there be a
roblem due to impurities by degraded bovine immunoglobulines?) and the
sample buffers contain phosphatase inhibitors (fluoride, pyrophosphate,
vanadate).

Did anyone experience this problem before? Any suggestions for a
remedy?

Your input is welcome!

Wolfgang

PS please reply to the group, the email ends in autotrash!



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