My protein of interest is in the cytosolic fraction of mouse amygdala (using an NE-PER kit) in western blot but in IHC it is mostly nuclear. Any ideas? Any idea for fractionation of frozen tissue with no cross contamination?
Yes I get band in nuclear fraction. And I get histone H3 band just in nuclear fraction but in very low level...I cannot use fresh tissue unfortunately.
no its not stronger :( I donno if its cross contamination or what because my H3 is just in nuclear fract. but this protein which is supposed to be in Nucleaar frac is in cytopl. frac and its not consistent with staining results...
Pooneh, Just curious if you ever got this protocol/kit working to your satisfaction? I'm looking to also separate cytoplasmic and nuclear fractions from frozen amygdala mouse punches but have no experience in these protocols. I've checked out the kit you listed above from Pierce, but it says that it works best for fresh tissues. Do you have any further insight?
Thanks!
Stephanie
Try to use a very gentle homogenization. Ideally your histon H3 signal should be very strong in nuclei even if you see some in cytoplasm. some cross contamination is not avoidable
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