problems with His tagged protein western blots
Posted 13 June 2010 - 01:46 PM
I am having problems with detection of my his tagged proteins on my western blots. I had no bands on my western even my his tagged protein ladder (which was supposed to act as my positive control) however staining of my membrane showed there was protein there. I know there was nothing wrong with the gel or transfer stages as a collegue ran their samples alongside mine and were fine. we used different antibodies: my primary was a tetra his antibody from qiagen my secondary was a anti mouse conjugate as required. I have trawled through the qiagen handbook and we followed the intructions to the letter, even my more experienced collegues are stumped, has anyone else used these antibodies and had a similar problem? Any ideas would be really well appreciated!!
Posted 13 June 2010 - 04:57 PM
Posted 13 June 2010 - 07:23 PM
We switched to Genscript's anti-his antibody and our problems pretty much went away (I am not affiliated with Genscript, or with any company, for that matter).
Posted 15 June 2010 - 07:07 AM
We have followed the storage instructions to the letter (in fact I got a little paranoid about it!)
No she used a different antibody. We did consider the fact the epitope was hidden in my protein, however the 6xHis protein ladder which should act as my positive control is not showing up, neither is a borrowed His tagged protein which has shown up previous western blots. I am still waiting for a response from them.