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Immunoprecipitation pH switch


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#1 Trof

Trof

    Brain on a stick

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Posted 05 July 2018 - 07:48 AM

Just a curious question.

 

Doing (co)IP for some time now, FLAG Ab with A/G agarose beads and now and then I get a sample, where the lysate (with 2x Laemmli, which is usually very blue) turns yellow. I had seen it in one of the sample types (FLAG only vector) in the past and now I see it in a sample that has contransfection of V5/FLAG tagged vectors, V5 one is a consitutively active mutant of a receptor. I do not take out the beads, just boil them with the lysate.

 

I boiled todays for 6 minutes, and I could see the yellowish beads on the bottom of the two replicates of the same sample type where I expect a lots of proteins. I try to pipet it up and down, but one of those still have some sticky stuff on the bottom, so I boiled it some more time (usually we use 6-10 minutes, IP samples are usually not very thick, so I do just 6) and it dissolved but turned yellow. These samples change back to blue color after loading to PAGE, and probably doesn't cause any problems that I would notice.

 

The color change is logically due to a pH change (somewhat below pH 4 if I rememvber correctly) but I wonder what is causing it. There should be only proteins in the IP, which denature after Laemmli addition. Or can it be some cofactors that get freed from a protein after that? DNA?? (FLAG tagged protein is DNA binding, but this buffer should not get nuclear fraction, theoretically).  What could get acidic or otherwise after denaturation of protein complexes? I have only limited knowledge of proteomics, so I'm curious if anyone of you knows.  Here, have a nice picture of my yellow sample in the meantime wink.png

 

IP pH.jpg  


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