Co-IPs and pulldowns of transcription factors - (Dec/15/2009 )

So, I'm trying to Co-IP or pulldown new binding partners to a specific transcription factor I'm studying (protein X). I've tried doing a few simple co-IPs, using S35 labeling and silver staining to look for bands that are unique to a co-IP with my protein X. I've tried transfecting protein X to overexpress it, then immunoprecipitating with an antibody against it, as well as using a myc-tagged version of protein X and a myc antibody. I haven't found any interesting bands yet, but I'm still continuing to optimize my protocol.

I just have a couple of questions:
1. How much does NaCl concentration affect protein-protein interactions in co-IPs? Because my protein is nuclear, many of the lysis protocols I have involve a high salt (400-500mM NaCl) step to break open the nuclei.
2. Another method I may try soon is using biotynalated beads and a target DNA sequence that my protein of interest is known to bind to. Has anyone tried using that method to pulldown binding partners of other proteins? If so, what buffers did you use?

Activating transcription factor 4 (ATF4/ CREB2) is a member of the.In contrast, pull-down assays using purified glutathione S-transferase.Interestingly, FIAT/ gamma-taxilin also interacts with the transcriptional co-activator.


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Velox on Dec 16 2009, 03:42 AM said: