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unstable flag tagged protein


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4 replies to this topic

#1 Lauretta77

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Posted 22 December 2011 - 02:54 AM

Hi
I used the expression vector pCDNA3.1/flag to subcloning the C-terminal region of BRCA2 and transfect it into BRCA2 deficient cells. After Immunoprecipitation and Western blotting analysis with a flag antibodies, I didn't see the expected band of approximately 80 KD but two bands of 50 and 30 KD.
Any suggestions?
Thanks in advance
Laura

#2 GNANA

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Posted 22 December 2011 - 03:20 AM

did you run the Antibody along with the IP, just to make sure your 50KD is not the Ab chain.....
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#3 Lauretta77

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Posted 22 December 2011 - 05:20 AM

No, I didn't! Thank you for your suggestion, I'll verify it.
Just an information, how many antibodies I have to run?

#4 GNANA

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Posted 23 December 2011 - 08:31 AM

i usually denature the same amount i use for IP.....may be u can also use bit less than that...
I would prefer being perfectionist rather than a passionist in Research.

I always had an alternate hypothesis....

#5 Lauretta77

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Posted 26 December 2011 - 03:29 AM

Ok!
Thank you very much!




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