CHIP with Tagged Proteins - (Oct/25/2005 )

Hi,

I need to work with tagged proteins and use antibody against the tag (FLAG or HA) to do CHIP. Anybody has any experience with CHIP against tagged proteins?? I really need help with this. I tried 3xHA and it doesnt seem to work. Also, tried using Triton based lysis buffer but still no band though I know the protein binds my target gene. I am going to try it a few more times with more starting material this time. Will also tag the proteins with FLAG and try CHIpping with Flag antibody but if somebody has some experience with this do let me know.

Thanks a ton!
Shail

just want to confirm some general aspects:
1: Does your prelimenary study show that the fusion protein (e.g. HA tagged) has biological function?
2: "no band" mean no protein been immunoprecipitated or no PCR band in enrichment test?
3. Besides the chip that you are currently doing, do you have any other experience on ChIP and get succeed?

Here are the answers to your questions. Thanks for trying to help.
S

1: Does your prelimenary study show that the fusion protein (e.g. HA tagged) has biological function?

-- Yes, I know that the full length tagged protein has biological function (atleast by IF colocalization with other proteins and by co-IPs).

2: "no band" mean no protein been immunoprecipitated or no PCR band in enrichment test?

-- no PCR band. Havent looked for protein on western after CHIPping. Was planning to check that.

3. Besides the chip that you are currently doing, do you have any other experience on ChIP and get succeed?

--- Yes I do Have been doing it for over a year now and it (usually) works fine...But this is the first time I am trying to CHIP a tagged protein and couldnt find a protocol for CHIPping tagged proteins in mammalian system.