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DNase,BrdU and GFP - (Jun/08/2001 )

Hi,I was wondering if anyone knows a simple method for staining cells with an anti-BrdU antibody, followed by a labelled secondary antibody, that does not involve the use of HCl (probably DNase). I would like such a method, because I want to stain GFP expressing cells and I expect that the HCl step in antibody detection of BrdU would possibly denature the GFP. Does anyone know otherwise?Thanks,Marty.


As much as I hate to say it, I have found that GFP is not a very robust protein. It doesn't last under extreme conditions (such as an acetone extracting cell membrane in preparation for antibody staining). My best suggestion for your experiment is to use a different fusion vector. You are already performing an antibody step, so you will get a stronger signal if your protein is fused to another tag, such as GST. Then use two different primary antibodies - the anti-BrdU and the anti-GST. I've had great sucess with the rabbit anti-GST from Pharmacia.
If you must use GFP, the anti-GFP antibody from Molecular Probes works well even on denatured GFP that has lost it's autoflouresence.
I don't think that was quite the answer you were looking for, but I hope it helps.

-Jonathan Sayer-