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Which technique is best for studying transcription factor-promoter interaction


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4 replies to this topic

#1 cells

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Posted 09 March 2011 - 02:53 PM

Hi, I was wondering if anyone would have any suggestion as to what technique would be best for simply studying if my androgen receptor which acts as a transcription factor is interacting with a specific promoter region within cells. I want to also know if, under different conditions, the interaction is stronger or weaker potentially leading to increased transcription. Would EMSA or ChIP be more convenient (and easier) to set up and perform? Is there any other way I can go abouts looking at this? Thanks for any feedback!

#2 pcrman

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Posted 12 March 2011 - 11:35 PM

Hi Cells,

You can do ChIP to show AR association with the sequence. ChIP is relatively simpler than EMSA. Nowadays, it seems ChIP has replaced EMSA experiment in many situation. By the way, have you found ARE in the sequence? if not, probably that is the first thing to do.

#3 laurequillo

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Posted 13 March 2011 - 12:02 AM

I agree with pcrman about the better quality of Chips results versus EMSA results. But EMSA is much easier than Chips. Sometimes you can spend months trying to optimize your chips, so maybe you could try both techniques in pararell.

There is another technique you can try (if you are not lucky either with Chips or EMSAS), that give you the same information than Chips but in vitro. You just produce biotin-primers spanning the region you are interested in your promoter, and amplify it by PCR. Then you incubate cell lysates with those purified fragments, and pull down them with Strep-Beads. And then you run a WB and develop against your target protein. It is like a Chip but in the other way around, and in vitro, like an EMSA.
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#4 cells

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Posted 13 March 2011 - 04:21 PM

Thanks guys...

pcrman, I'm actually planning on looking at known ARE so that shouldn't be much of a factor.

laurequillo, the second technique you mentioned sounds interesting but I actually need the results from this experiment as soon as possible. If I know sequence of the response element and I use a commerical ChIP kit would the process be relatively easy?

#5 laurequillo

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Posted 23 March 2011 - 12:49 PM

yeah, Chips are not exactly easy!! :P But It will depend of your antibody, your primers...
I think EMSA is an easier technique (not better, but easier), , but of course you can try the Chips and if not do the EMSAS.
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