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problems with His tagged protein western blots


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#1 BB25

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Posted 13 June 2010 - 01:46 PM

hi,

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!!

Thanks

#2 bob1

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Posted 13 June 2010 - 04:57 PM

Obviously the problem is with the antibody you used. They do go off frequently and should be stored frozen at -20 deg C in aliquots and thawed for use once only and then kept at 4 deg C for a couple of months at most.

#3 HomeBrew

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Posted 13 June 2010 - 07:23 PM

We had problems with several vendor's his-tag specific antibodies (Invitrogen, Qiagen, etc.) -- sometimes, on particular proteins, they just don't work (see, for example, Qiagen's panel here). It's unclear from your post whether your Ab detected your colleague's protein (which I assume is not the same as yours) -- did it? If your Ab detected your colleague's sample and not yours, the problem may be that your epitope is hidden. If it detected neither protein sample, maybe your antibody (or your secondary antibody) has gone bad. What does Qiagen say about the problem?

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).

#4 BB25

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Posted 15 June 2010 - 07:07 AM

Hi,

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.




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