Western blot does not match IF - (Apr/03/2012 )
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
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.
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?