transcription factor ChIP - High signal in no Ab control (Jul/05/2005 )

Hi Guys ,

Does anyone ChIP transcription factors? I've been trying for several months to ChIP the transcription factor SREBP-2 from THP-1 macrophages. The one biggest problem is the background. There seems to be alot of non-specific binding to the protein-G sepharose beads that i am using. I've tried longer pre-clearing and extra washes, but i don't get much of a reduction in background.

I'm following the Upstate ChIP protocol except that i am using sepharose and not agarose.

I've read about pre-blocking of the sepharose beads before use. Is there a protocol on how to do this?

Does anyone have experience with something similar? or is there a protocol out there for specifically chipping transcription factors?

Just so u guys know, i use 1ul of chipped DNA for PCR, 35 cycles of 94 deg 30s, 58 deg 15s, 72 deg 15s. Primer Tms are 58 degrees.

babynekko

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hi,
why not reduce PCR cycles? linear amplification?!
Some ChIP data shows " black &white "( all/ nothing)
picture has lower/optimized PCR cycle numbers.

have u any positive Ab control( ChIP proved ) ?

BTW,I'm trying TF ChIP too. we could make progress together
with communication in forum ?!
Good luck!

ZY

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Thanks for replying

I'm curious, how many PCR cycles is the "recommended". The reason i use the high cycle number is that the antibody i am using is notoriously bad. Problem is, it's the only antibody available.

I have tried a positive control, but i'm not sure if it has worked or not. The background for the negative control was about the same level of intensity (gel pic) so it may have just been non-specific stuff that i amplified.

Do u have a problem with non-specific binding?

And yep, progressing together would be great! Someone to share the experience with!
nekko

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Hi all, I also get background with the no-antibody control (also using the upstate kit), I agree with zy101 reduce cycles etc. to reduce the appearance of background bands. Upstate reccommends using only 20-25 cycles and doing two rounds if necessary, in order to keep nucleotides in excess. I am planning on trying this next, perhaps the difference +/- antibody will be enhanced.

Also, we have included an additional centrifugation at 15,000*g after the overnight incubation with/without antibody (but prior to adding the beads) this will help in case things are precipitating out of solution during the overnight incubation.

BTW-----If you look at the new active motif kit their gapdh positive control shows the same background, indicating that this may be a normal thing.

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I have seen a protocol using 1mg/ml yeast tRNA as blocking reagent.
Another choice is to use a more stringent washing buffer.

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I'm a bit curious about the blocking agent. I've seen many protocols use salmon sperm DNA, but some protocols also use BSA + salmon sperm DNA. How do these actually block the beads?

babynekko

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