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Overexpressed protein shows a band higher than expected?!


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#1 Ambinlab

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Posted 12 August 2012 - 12:29 PM

Dear all,

I am trying to optimize an antibody for a tumor suppressor. I tried another commercial antibody which gives the same background as my own antibody. The problem is when I overexpress the protein in HEK293s, it shows a big band on ~10kda higher than what I expect the band to show up (~48kda). I even used HAtagged plasmid to overexpress and probed against HA. The same band showed up (I mean it was 10kda higher than what I expected).

Should the overexpressed sample always show the same band size as endogenous? Or is it possible that the overexpressed sample runs differently?

Thanks

#2 bob1

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Posted 12 August 2012 - 12:52 PM

It is very common for proteins not to run at the expected or predicted molecular mass. In denaturing systems, the overall charge of the protein still has some bearing on how it runs, despite the SDS and supposedly linear conformation.

It is also possible that the protein is post-translationally modified in some manner.

Antibody data sheets can also be a bit misleading - often they show bands that are against the antigen only, or against purified protein that was expressed in a non-native system (e.g. e. coli for a eukaryote protein).

#3 klenow

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Posted 22 August 2012 - 02:02 AM

I think the question is if with these antibodies you detect that the endogenous protein has a lower molecular weight band. If this is not the case and you never detect endogenous levels, it is not surprising to have differences between the stimate MW and the real one. To be sure, you can check the overexpression construct to make sure the sequence is correct.
If everything is OK and the endogenous protein does have a lower MW could be that, as bob1 suggested, overexpressed protein is having some posttranscriptional modification... however, I would try to do RNAi against your protein of interest, because maybe the antibodies are not sensitive and/or specific enough. I had a similar problem, but the difference between real and expected MW was too much to have doubts. The antibody only recognised overexpressed protein (single band) but when non-transfected cell extracts were used, the antibody recognised a non-specific band... Just to be sure I performed RNAi against that protein and confirmed the "endogenous" protein recognised by the antibody was not the good one...

Edited by klenow, 22 August 2012 - 02:03 AM.





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