Western blotting and immunofluorescence. Results don't match. - This is madness... (Jun/22/2007 )
I have detected a protein in cytoplasm, while all papers say it should precipitate in chromatin. I can't see it in nuclear extracts, while cytoplasmic look just fine. I have repeated the procedure, so the samples aren't switched... Is it possible that the protein is visible in nuclei in immunofluoresence, but not detected by western blotting?
Is it me, antibody or the papers??
I'm stunned.
Is it me, antibody or the papers??
I'm stunned.
You can see it in the nuclei but not cytoplasm using IF but cannot found in nuclear extract by WB?
How did you do the extraction? Maybe the cytosol got contaminated with lyzed nuclei. What about positive controls for each fraction? Do you found other nuclear proteins in the nuclear extract but not in cytosol?
Is it me, antibody or the papers??
I'm stunned.
You can see it in the nuclei but not cytoplasm using IF but cannot found in nuclear extract by WB?
How did you do the extraction? Maybe the cytosol got contaminated with lyzed nuclei. What about positive controls for each fraction? Do you found other nuclear proteins in the nuclear extract but not in cytosol?
I'm getting to it. I'm reprobing the blot right now. The IF was done not by me, but by another lab. It's supposed to act with the chromatin under certain conditions (which I duplicated), so it's really weird I can see a strong band in cytoplasm, not in the nuclei.
Then let see how the positive controls look before being stunned
Also, I would suggest that next time try to reproduce the reported result (the IF in this case) first. I would still repeat the IF anyway if I were you Then let see how the positive controls look before being stunned
Also, I would suggest that next time try to reproduce the reported result (the IF in this case) first. I would still repeat the IF anyway if I were youHuh...
Now it gets weirder, the histones are in the nucleus as it should be, not in the cytosol, another nuclear protein is in both fractions (well, it's not a control really, it's mostly nuclear but could be also in cytoplasm, I was just curious).
I'll have to do the IF thingy, might be tough, we don't have the ingredients. Argh.
Is it possible that IF is more sensitive and there is some of my protein inside the nucleus, but too small to detect it by blots?
hi,
I have several points that may help:
#1 IF results can be easily manipulated. You should do the IF or immunohistochemistry yourself.
#2 Some proteins can be found in the nucleus and/or the cytoplasm depending on the cell type. Protein localization can vary widely be tissue type and especially between cell types when using cancer cells.
#3 When looking at nuclear versus cytoplasmic fraction use controls for BOTH (!!) cytoplasmic and nuclear fractions. There are several that can be used. Look through the literature. If you have proper controls the problems become clearer.
#4 When isolating nuclear versus cytoplasmic extracts it is easy to cross contaminate fractions so controls in #3 are impt.
Good luck!
Then let see how the positive controls look before being stunned
Also, I would suggest that next time try to reproduce the reported result (the IF in this case) first. I would still repeat the IF anyway if I were youHuh...
Now it gets weirder, the histones are in the nucleus as it should be, not in the cytosol, another nuclear protein is in both fractions (well, it's not a control really, it's mostly nuclear but could be also in cytoplasm, I was just curious).
I'll have to do the IF thingy, might be tough, we don't have the ingredients. Argh.
Is it possible that IF is more sensitive and there is some of my protein inside the nucleus, but too small to detect it by blots?
Thank you all very much. I've read up more and the overall weirdness of these results is starting to be interesting. Not judging prematurely of course :>
test for the purity of your nuclear fraction with e.g. Lamin B1 and for the cytoplasmic fraction alpha-tubulin. Check your western blots with antibodies against these proteins.
I hope that this suggestion will help.