Problem with IP / Immunoprecipitation Assay

Jens Wuerthner, M.D. wurthnej at dce41.nci.nih.gov
Tue Oct 19 12:36:37 EST 1999


Hi Everybody,

I got a strange problem with my IPs:
After Lipofectamine transfection of COS-1 cells (plated at 1.5 x 10e6
per 100 mm plate) with different epitope-labeled constructs, I used to
see an interaction of certain proteins that I am interested in. Suddenly
things stopped working, no more interactions. I am now back to
interaction of my positive controls, but not other proteins that
previously did interact.

I use a lysis buffer with a final concentration of NaCl at 150 mM, 50 mM
NaF, 10% glycerol, 5 mM EDTA, 25 mM HEPES (pH 7.5) and 1% Triton X-100.

Intermittently I got some interactions with a lysis buffer containing
half the NaCl, but also that buffer lost its magic. Also tried Tris
instead of HEPES, no difference.

If anybody has some ideas, please post them.

Thanx.

Jens Wuerthner
LCRC
NCI/NIH
Tel: 301-594-5729
Fax: 301-496-8395
wurthnej at dce41.nci.nih.gov




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