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His-tag purification and his-antibody for western - (Jan/10/2002 )

Dear all
I met a problem in purification my protein expressed from pichia KM71 cell. it is a novel membrane protein and I can detect the expression in the crude membrane of km71. After purification with the Qiagen Ni-NTA kit, I got a very sharp band, a little smaller than the expected size. To my surprise, the band can not be detected with anti-His in Western. WHY??
Hoping get some information from any one.


I think, may be, that HIS sequence could be eliminate during expression.
You can see protein after purification with coomassie but not detected with anti-HIS antibody.
good luke


do you make western blot at native conditions or denaturent conditions, hence if you make it in native conditions and if histag was stucked in the protein, it may not be detected. And are you sure that your second anribody recognizes primary antibody, you can test this step or may be you shoul optimizze western blot conditions....