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Western blot does not match IF


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2 replies to this topic

#1 sBrow

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Posted 03 April 2012 - 12:42 PM

Hi please help if you can
I have seen two posts on here with this problem, but the answers have not helped so far...

I get nuclear protein (about 90%) with immunofluorescence, but using the same antibody I get 95% cytoplasmic protein in western blot (NE-PER kit). My extractions are very clean, i have used antibodies to measure this.

The only thing I can think of is that SIRT1 my molecule of interest is somehow leaving the nucleus during the extraction.

I am going to try cross-linking with formaldehyde, and glycine.

Can anyone shed light on this?

any ideas would be much appreciated
SB

#2 Curtis

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Posted 03 April 2012 - 05:12 PM

Have you checked if your IF antibody can be used in WB too? Some antibodies don't. It could be that you are seeing non-specific band in your WB. I had this problem. My anti-Bax 6A7 worked well in IF, but didn't lable a thing in WB.

#3 sBrow

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Posted 03 April 2012 - 10:31 PM

Yes - the product sheet says it should work for both. thanks for the thought...keep them coming!
I'm going to try crosslinking before extraction, has anyone used this?




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