Immunostaining of cytoplasmic proteins in nucleus, why? - (Jan/02/2007 )
We have experiencing an artefact staining of various cytoplasmic proteins in nucleus. We use 4% PFA (in PBS) fixation step with following permeabilization with TX100 and ordinary double staining with primary and secondary antibodies. Assuming that antibodies are specific, what could be an other explanation of our unusual observation of intracellular localization of cytoplasmic proteins in the nucleus? What are the hints to avoid the problem?
Thanks to anybody for the suggestion and advice!
Mindo
may b these proteins also localise to the nucleus under normal or under specific circumstances.
Or if u think there is problem associated with immuno staining, u could try lot of dif. things.
like lower conc. of antibodies (primary and secondary), block longer and try dif. blocking serum. wash additionally.
ofcourse, an unwanted staining or background is usually due to antibody specificity, primary and secondary.
AB are specific, protreins are not localize in the nucleus acording the cell fractionation and literature. We observed the phenomenon with at least 5 AB (including mABs). Pitty, but we've tryed methanol fixation, titrated antibodies and nothing work - nucleus almos all the time is pedominantly stained.
Many times I find it difficult to believe literature on immnuostaining data as its somehow not reproducible.
Write to a coauthor of a paper showing this immunostaining and ask for any particular step to get the desired result.
As I wrote earlier, there r many things one could tr different in immnuostaining.
Good Luck !!!
Thanks to anybody for the suggestion and advice!
Mindo
did you make a XYZ-scan to make clear that your protein of interest is actually found in the nucleus?
some cytoplasmic proteins such as calmodulin are also abundant in cytosol, but larger cytoplasmic proteins should rarely be found in the nucleus
Thanks to anybody for the suggestion and advice!
Mindo
did you make a XYZ-scan to make clear that your protein of interest is actually found in the nucleus?
some cytoplasmic proteins such as calmodulin are also abundant in cytosol, but larger cytoplasmic proteins should rarely be found in the nucleus
YES we did Z-scan, and the proteins are above 70 kDa, and they dominate in the nucleus, that is our problem.
Are you using a retrieval step? Because sometimes over-retrieving the tissue causes faint "staining" in the nucleus.
we work with tissue cultures only, so I guess we do not perform retrieval step.
Have you tried to stain cells without primary antibodies? May be this stuff recognizes smth in the nucleus?
Nucelus staining comes from primary antibodies, and this problem comes not from one AB...