Immunohistochemistry - Leakage of protein from nucleus?

Melissa Greeve gliftor at hotmail.com
Thu Oct 4 03:44:54 EST 2001


Hi everyone again,

Thanks for your replies to my last posting of immunoprecipitation
woes. I seem to have managed to get IPs to work, resorting to the use
of a more dense SDS-PAGE gel to avoid the problem of the protein G
band - I have, however, yet to get consistent results. But it's only a
matter of time, more sleep deprivation and some more tweaking... :)

Anyway, onto my next curious problem. One of the important experiments
that will (hopefully) nicely supplement the IP results will be
immunohistochemistry of cells grown on slides. The protocol itself
appears to be fine, but one of the proteins I have been staining for
is meant to be (published for other cell types) nuclear - but I have
only been able to show that it is cytoplasmic and perinuclear. So far,
I have tried two different antibodies to the protein. In contrast, I
have shown that another protein, also published as nuclear, is indeed
expressed in the nucleus and cytoplasm in a cell-cycle dependent
manner.

Someone has suggested to me that one of the potential reasons for this
discrepancy could be leakage of the protein from the nucleus into the
surrounding cytoplasm during insufficient fixing of the slide. So, I
tried chilled acetone, with nasty results - the cells, understandably,
dehydrated.

Is this a logical explanation? Why would one protein act like this
when fixed in 95% ethanol and not the other (both have similar
cellular functions)? Can a PhD get any more complicated??

I will try a different cell type as a control, to confirm that the
apparent localisation of the protein in cytoplasm results from
insufficient fixing, otherwise, maybe my cell type is a strange
exception! It's important to demonstrate the true localisation of this
protein before I obtain any results (of course, analysis of this
protein is crucial to my project!)

Thanks again,
Melissa Greeve
PhD Student
University of Western Australia




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