Using denaturing protcol for his-protein purification - Can it still be used for antibody production????? (Oct/18/2006 )

Hi there -

I am having some problems with getting other proteins binding to my ni-nta superflow resin (qiagen) column. I am using the native protocol. My his-protein is being bulked up in ecoli BL21.

Before I start making changes like increasing imidazole in washes, adding tween, adding BME, using different concen. of imidazole in elution steps, etc. I was wondering if I could just use the denaturing protocol instead. I told the tech at Qiagen that I wanted to make antibodies from this protein and they said that the denaturing protocol would probably work just fine (?) I wanted some feed back before I go through the effort.

Thanks,

R

Hi -

Thanks for your reply. Actually my protein is binding to the column along with unwanted proteins. I wanted to use the denaturing protocol instead of the native protocol because it is more stringent. However, by using the more stringent denaturing protocol will I still be able to raise "good" antibodies against it???

Thanks,
R

You can raise polyclonal antibodies to denatured proteins. You might not produce antibodies that recognize a three-dimensional epitope that forms when the protein is properly folded, and you may produce some antibodies that fail to react against the properly folded protein, but there will still likely be many other antibodies in the polyclonal population that will react with the native protein.

holyrail,
I am using pQE31 (Qiagen) trying to express my protein in BL21. But I got proein expression both in the IPTG induced and non-induced group. The pQE31 doesn't have the lac repressor coding region, so it need to be used with a E.coli strain which can express lac repressor. Will BL21 do this job? Thank you very much!
--sure