HIs tag recognition problem - Ways to expose the HIS tag (Nov/16/2007 )

Hi,
I'm having problems to detect a 35kda protein (a pentamer) in WB with the anti HIS antibody. The antibody works because it reacts a lot with the marker so I suppose the marker was purified with His tags (rpn800, GE).

I've tried different antibody dilutions and I kept seeing nothing in the WB upon induction of IPTG to produce my protein (encoded in a PQE70 vector from Quiagen).

Any ideas?.

I've only tried native purifications of the protein but recently I did a denaturing purification but instead of seeing a 35kda band in the coomassie, I saw several bands in the eluates.

I'm supposed to get big quantities of this protein (in the mg range) any ideas or tips for the large scale production? I've never made a large (10l) production before.

Best regards,
Carla

Dear Carla


As i undestand ur using pQE-70 vector with N-terminal His-tag. This kind of vector should act as positive control for ur protein expression.

You wrote that u didnt get any protein (in W. under native condition purification, meaning there was problem with the translation of ur gene and no HIS-tag was

form.

Check again ur sequence and look for stop codon or mis-match with the sequence.

The bands that u got in the gel under denaturing condition, are probably bacteria proteins (heat-shock protein or cellular protein) form during the

overexpression process.

Best regards