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Unexpected expression of my protein in HepG2


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#1 VICKY01

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Posted 21 May 2011 - 08:30 PM

My protein was never expressed by HepG2 cells. This was concluded after several attempts at detecting the protein by Western blots. This was tried by my lab (several members) and another lab. Recently, I started detecting this protein via westerns which is quite alarming. I am wondering if it has anything to do with the cell line source. There is no documented evidence for the expression of this protein in HepG2 cells. Are there any possible reasons for the sudden expression of this protein?

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Vicky01

#2 bob1

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Posted 22 May 2011 - 05:57 PM

Is the antibody you are using a new one for the same protein? How have you confirmed that this is the protein you think it is on your westerns (e.g. by peptide competition)?

#3 VICKY01

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Posted 22 May 2011 - 08:17 PM

Is the antibody you are using a new one for the same protein? How have you confirmed that this is the protein you think it is on your westerns (e.g. by peptide competition)?



Yes, I have confirmed it by treating the cells with a specific inhibitor of the protein..the expression level dropped. Plus, I run a positive control which showed up at the same molecular weight. No change has been made to the antibody used. Different aliquots of the same Ab stock.

#4 bob1

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Posted 23 May 2011 - 05:07 PM

In that case, it looks like HepG2's express your protein. Have you or others looked for RNA too? Is it possible that the cultures you are using are old or contaminated, thereby altering their expression?

Absence of evidence is not evidence of absence.




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