Perhaps since it is a new antibody, like Rafal said, this may not be working in the same manner. Perhaps maybe a wrong antibody was ordered, or it has been changed, which often happens. Make sure that this antibody is suitable for ChIP assays.

Check the size of chromatine fragments and if the Ab is suitable for ChIP assays. Maybe include an antibody as positive control to check if your solutions and your crosslinking was all right.

Hi Anahit, I hope everything is fine. Where are you now? Normally the most problematic steps in the ChIP is the cross-link, the chromatin fragmentation and the antibody. The most obvious problem can be the antibody is not good for the native protein, but maybe you change other things like the sonicator or the vacuum pump so you need to adjust the conditions again.

Hi there, in addition to all what they have mentioned, first check which agent to use for crosslinking and weather to use single or double fixation. then, optimize your sonication step. once u have the best condition of sonication for different crosslinkers, try to use different ab for IP, different concentrations and also different durations of incubation with ur ab.

I don't know weather you have no DNA at all after DNA purification or there was no enrichment. moreover, snication condtions are sensitive o the numberof ur samples and their volumes as well. best of luck :)

Hi all, thanks for all comments SO this is the situation:

-sonication is fine, since I have check the samples on a gel before and after sonication and I get a nice smear from 200-700bp

- the input samples are also just fine

-the antibody should be fine as well, since I used it before with similar samples 8 months ago. I orgeres a new aliqout but it should be just fine, its ChIP grade anti-GFP from abcam.

-my problem is that when i do a regular PCR to test the Chips samples before qPCR, I get nice bright band in input, faint band in -AB and very little or nothing in +AB.

My solutions my get bad, since they are almost 4 months old, I feel like the elution step i not working therefore my antbody+protein comlex stays on beads and i get no DNA in the solution.

All these is very strange since the same protocol was working fine for similar samples last summer and when i got back doing ChIPs nothing works. I am also using low-retention tubes just in case my antibody sticks on plastic.

I am going to make up new fresh solutions and try with a single sample....hopefully it gets to work...fustrating!!!

Try to set up sheared chromatin + Antibody rxn for 2-3hrs at 4degree and then add beads, incubate o/n. You can use magnetic bead based kits from Active motif or Magna ChIP™ Millipore. I regularly use them for my ChIP studies, no problems so far.

Hope things works out for you!!!

Novel AHP antibodies available at: http://www.protean.cz/en/recombinant-protein/51/anti-ahp-panel