co-IP problem - (Aug/04/2005 )

I'm having trouble performing coIPs--
Have been trying to determine whether protein A
interacts with protein B.

Previously it has been shown that protein A
interacts with another protein, C.

I cannot reproduce the A-C interaction,
and hence am unable to determine whether
my negative data for A-B is real or just
a problem with the assay--
I need to get a positive control to work
before I can accept a negative result.

I also have not been able to reproduce
a separate interaction, D-E. While it is possible
the previously published interactions were not
robust results and the authors themselves could
not reproduce them reliably, it seems unlikely
that these two independent binding interactions
in the literature would fall into this category since
the data themselves in the figures look good (?)

I am transfecting 2ug of each plasmid into 293T cells;
my transfections work, as I am able to detect
the overexpressed tagged proteins by western blot
in whole-cell lysates; when I IP for protein A,
run out the IP on western and blot for protein A,
I see a strong band; so I apparently can IP protein A
successfully, but I just cant detect ANY binding
partners (B or C). I use 300ug protein lysate per IP.
I incubate lysate with both beads and primary antibody
simultaneously at 4 degrees with rotation O/N.

I'm wondering if I'm encountering a simple technical
problem with my technique, or with my buffers;
I used standard RIPA buffer (see below), while other
groups used less harsh lysis buffers...and included
magnesium and/or zinc in some cases.
BUT, ONE study has actually already demonstrated
the A-B interaction in cos7 cells, and they used
standard RIPA buffer


Thanks very much for any HELP (!!)



Here are the previously published data and buffers:

**MY LYSIS BUFFER**
RIPA BUFFER
150 mM NaCl
1% NP-40
0.5% deoxycholate (DOC)
0.1% SDS
50 mM Tris pH 7.5
ddH20
PROTEASE INHIBITORS
NaF
NaOrthovanadate

1. protein A-protein B (PUBLISHED)
cos7 cells
500ug lysate per IP
add beads POST-Ab
RIPA
140mM NaCl
1% NP-40
0.5% DOC
0.l% SDS
20mM sodium phosphate pH 7.5
4mM NaF, 10ug/mL aprotinin,10ug/mL leupeptin, 10ug/mL PMSF,
0.1mM NaOrtho, 10ug/mL glyercol phosphate

2. protein A-protein C (PUBLISHED)
293T cells
transfect 6ug DNA of each plasmid
lyse 20 minutes on ice with 1 million cells/100ul extraction buffer
150 mM NaCl
0.5% NP-40
50 mM Tris HCl pH 7.6
1 mM MgCl2
50 uM ZnSO4
1X EDTA–free protease inhibitor cocktail [Roche]
10 mM NaF, 1 mM Na3VO4

3. protein D-protein E (PUBLISHED)
293T cells
lysis buffer
100 mM KCl
0.1% NP-40
20 mM Tris-HCl [pH 8.0]
5 mM MgCl2
10% glycerol
supplemented with freshly prepared protease inhibitors.

use a different lysis buffer.. RIPA is a strong buffer with lots of detergent and SDS, which is likely disrupting all protein-protein interactions in your sample. You can try a basic NP-40 - based lysis buffer (like whats listed in your protein A-C section), and i've had great co-IP results using a commerical lysis buffer (M-PER) from pierce, too. RIPA is definitely too strong for co-IP