Co-immunoprecipitation of nuclear proteins

jstrassw at OPAL.TUFTS.EDU jstrassw at OPAL.TUFTS.EDU
Fri Feb 7 09:52:42 EST 1997


Hello!
Well, you might have run into a biochemical dead end.  Try gently lysis
(even freeze thaw of tranfected cells in PBS- you might be surprised that
your proteins magically come out).However, you might forget the Co-IP
stuff because these days you need some functional data to get your paper
puiblished anyhow.  You might also try genetics:   fins a mutant in your
"bait" that doesnt bind your new cDNA and see if it bahaves like the
wild-type protein.

Good luck,

John
ps  are you getting a good tranfection eficiency in your COS?


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John Strasswimmer, MD,PhD Candidate    | Phone (617) 636 8396
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On Wed, 5 Feb 1997, Richard Dahl wrote:

> 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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