ChIP troubleshooting - controls for each step? (View forum version)


Posted 30 October 2012 - 07:45 AM

Hello everyone,

I have unsuccessfully tried to chip a potential overexpressed flag-tagged TF and since that did not worked
I have now started to try to chip Histones H3K9me3, but that does not seem to work either.

So something in my method has to go wrong. I was thinking of performing the same protocol again and
try to control which step gives me trouble.

I already checked the fixation by adding closslinked, decorsslinked and "decrosslinked + prot K digested + colum purified" material on
a agarose gel. I can see the crosslink and I can see how it is reversed by my elution buffer. And the fragment size is between 100-500 bp. So this step works.

Today I put crosslinked, decrosslinked material and whole cell lysate on a western to see if the antibody still recognizes the histion.
Could I destroy my epitopes by sonication?

How would you controll for the washing steps etc.? Would you also load that material on a Western?

I use a standard protocol which is highly similar to the magna ez protocol from millipore.
My aim would be to get a detectable amount of DNA that is significantly higher than in my control.
I use the Qubit Flourometer detection system which works similar to the picogreen kit and only measures dsDNA.
I can't do qPCR, at least not with my later protein of interest, since it has not been mapped to the chromatin yet.
This is why I will have to relay on the amount of DNA I get.

I tried Dynabeads and they also give me a DNA background (same amount as my sample atm, but then again the method is not
even working with histones). So would you also have sugesstions as how to get rid of the background?




Posted 01 November 2012 - 01:28 PM

I would do the K9me3 ChIP (whose enrichment profiles have already been determined) and ChIP for a TF with known binding NFkB or CREB..................and do the qPCR...........if it work then ChIP with your TF should work in theory, as long as your tagged TF is expressed as desired...........also consider using M2 coated agarose or magnetic beads.


Posted 01 November 2012 - 09:04 PM

Sometimes tagged exogenous proteins may not behave the same as endogenous ones such as DNA binding.

How many cells do you start with? If the signal is weak, you may need to increase the starting number of cells. This strategy works very well for us.


Posted 02 November 2012 - 01:34 AM

Thanks a lot! I will try that.