Hallo everybody!
I have got big trouble in purifying p65-GST and p50-HIS-Tag proteins.
After expression in E. coli and isolation on gluthation-sepharose and
talon-resin, respectively, I got both proteins. On SDS-PAGE are some
impurities visible, which are mostly due to copurification. Therefore I
tried a lot of additional methods for a second purification step
(gelfiltration, IEX-HPLC with anion and cation-exchanger,
calf-thymus-DNA-columns, Hi Trap SP Sepharose columns).
But in all cases I got the same phenomenon, the proteins did not bind to
anything, they always came down in the void volume. I tried a lot in
changing the parameters, like isolation with or without 1% Triton 100,
with RNase/DNase, with beta-ME, with DTT, different buffer-systems and
different pH-values. I tried urea in different concentrations, but in
all cases I got no specific binding to any material.
In my opinion, it is due to the assembly of huge complexes. But I do
not why I am not able to break them down. The only success was the
reducing of impurities by isolating p65-GST with 500 mM NaCl, but this
did not change my basic problem.
Therefore I read a lot of postings in this newsgroup and saw, that
this is a common problem, but I could not find a specific solution for
it despite of playing around with all the parameters and waiting for
good luck. But I have the impression that there is something basically
going wrong. Despite of all things I tried I did not even get a hint of
success.
Thus I hope that there is somebody out there, who has an idea of
what is going wrong (sorry, I can not summerize all the methods, it is
too much), so that I do not go crazy and depressed by my stupidity.
Thank you for reading this long posting,
Alexander Drung
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