3 replies to this topic

[cathyolive]

I have identified a 70bp region of interest within my plasmid enhancer which is important for high-level transgene expression after in vivo transfection into mouse lung.  My transfection efficiency is very low so ChIP is not really an option.  I would like to further identify which TFs are binding to this 70bp region of interest; I have a putative list of binding sites to get started with.

I'm looking into EMSA or DNA pull-down assays but am struggling to understand how I can do this and would appreciate any feedback.

My components are:
mouse lung nuclear extract
70bp region of interest from my enhancer
transcription factors

Do I mix the nuclear extract and my linear 70bp sequence and then add my TF probes?

No one in my lab does these assays so any help would be much appreciated.

[pcrman]

I think 700 bp sequence is too long for EMSA and even for footprinting. If you use such long sequence for EMSA you could get many binding by other proteins or non-specific binding which will retard the migration of your probe. I would suggest that you first do reporter assay to narrow down the responsible sequence by serial deletion.

Once you have ~300 bp target sequence, you can do footprinting to the protected (bound by proteins) region. Then design oligos (~50 bp) for the protected regions and use it as EMSA probe.

If you have further questions, don't hesitate to ask.

[cathyolive]

Thank you, but my target is 70bp, not 700bp

[pcrman]

I am sorry. Then 70 bp is good for EMSA. You need to synthesize two complementary strands of the sequence and make them a dsOligo probe. Nowadays, non-radioactive methods are a commonplace although as as sensitive as radioactive one. You also need a control probe containing scrambled sequence.

You said   "linear 70bp sequence and then add my TF probes". What do you mean by TF probes?