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I can detect my Flag-HA tagged protein with anti-HA but not anti-Flag

Western Blotting Epitope Tag HA Flag Cloning

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#1 Epigeneticist

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Posted 02 May 2014 - 09:13 AM

I have a human protein that is tagged at the N-terminus with a 1x flag tag followed by an HA tag. After transient transfection of this vector into 293Ts and protein harvest, I can detect the protein by western blot with an anti-HA antibody but not the anti-flag antibody (Sigma Flag M2). Has anyone had issues with a 1x flag tag? I know the anti-flag antibody works because I can detect another protein that is flag tagged. 

 

Kozak - 1xFlag - HA - ORF

 

Any idea what is going on here?

 

Thanks!



#2 Curtis

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Posted 02 May 2014 - 11:20 AM

I stopped using M2 long ago because of such problems. I couldn't use it in IP experiments because it was too sensitive to detergents in my lysis buffer.

#3 Missle

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Posted 05 May 2014 - 07:53 AM

I've just started using M2.  What antibody do you use now?



#4 Epigeneticist

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Posted 05 May 2014 - 06:12 PM

I've just started using M2.  What antibody do you use now?

 

I am sure your question is in reference to curtis but I thought I would give some feedback as well. HA tag and anti-HA works well for me. This was the first time trying Flag and M2. 



#5 Curtis

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Posted 06 May 2014 - 02:13 AM

I dont use Flag anymore. His works better for me. I think Sigma-Aldrich introduced a new Flag system already called 3x. Im not sure if they are still selling the original Flag. I think only Sigma has the right to sell this. Because I never saw other companies sell it.







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