How can I troubleshoot this STAT1 homodimerization assay?

09 September 2018 0 5K Report

I am trying to detect STAT1 homodimerization by first transfecting 293T cells with 2 STAT1 constructs, one with a GFP tag and the other with a FLAG tag, and then treating cells with either IFN-beta (1000U/ml) or IFN-gamma (20ng/ml) for either 1 to 4 hours. Next I lysed the cells with NP-40 buffer and performed a typical CoIP protocol with FLAG-beads. In my elutes, I can detect FLAG-tagged STAT1, but I do not see any of the GFP-labeled STAT1.

Am I using the wrong stimulus and/or cell line to induce STAT1 dimerization? Or could it be the location or identity of the tags? Both the GFP and FLAG-tagged construct are tagged at the N-terminal of the STAT1 construct.

Any advice on how to troubleshoot this would be much appreciated. Thank you.

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