Co-immunoprecipitation of nuclear proteins

Richard Dahl rdahl at mindspring.com
Wed Feb 5 21:53:43 EST 1997


I am hoping that someone might have some advice on different conditions
for attempting to co-immunoprecipitate two nuclear proteins from mammalian
cells.  I recently isolated a novel nuclear protein using a putative
transcription factor as bait in the two-hybrid system.  Although I can
demonstrate interaction in vitro I so far have not been able to
demonstrate an in vivo interaction.  I have co-expressed both the full
length target and bait proteins in COS1 cells and have attempted to
co-immunoprecipitate from lysates prepared with three different lysis
buffers-- RIPA, NP40/DOC and a Brj97 containing lysis buffer.  I can never
see a co-immunoprecipitation above background levels of non-specific cross
precipitation using any of these lysis conditions.  I have done this both
as an IP western and with 35S labled cells.  

By immunofluorescence I can see some overlap of expression in the nucleus
with confocal microscopy however the overlap is variable from cell to cell
and localization is never exactly identical.  Both localize predominantly
in speckled patterns perhaps suggesting some association with nuclear
structures.  Using a cellular fractionation protocol that I saw in a paper
demonstrating PML association with the nuclear matirix, I see that both
target and bait proteins seperate into the 2M NaCl resistant pellet that
supposedly represents the nuclear matrix.  So my guess is that perhaps the
interaction occurs in these potentially insoluble nuclear structures and
so far attempts to I.P. have either not disrupted these structures to
release my proteins of interest or disruption of these structures also
disrupts the interaction.

Anyway if anyone has any lysis conditions to suggest or any potential
protocols that might be worth a try I would appreciate the help.

Rich



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