I just performed a ChIP experiment following the exact protocol as I have successfully used before. After ethanol precipitation I see a large white pellet in all my ChIP samples, but not in the input.

experimental details:

cell line: human cancer cells

amount of cells: 10-20 million

amount of antibody: 10-20 ug

shearing method: bioruptor sonication in buffer with 1% SDS, then diluted 1:5 to dilute SDS

- following IP, beads were washed (low salt, high salt, LiCl ,TE buffers) then chromatin was eluted off the beads in 200 uL elution buffer (1%SDS, 0.1M NaHCO₃)

- input was made up to 200 uL in H2O

- cross-links reversed and proteins digested overnight 65 deg by adding 8 uL 5M NaCl and 2.5 uL proteinase K (20mg/mL)

- RNAse treatment 37 deg 1 hr by adding 5 uL RNAse A (4mg/mL)

- phenol-chloroform-isoamyl extraction (using Phase lock gel tubes, so no interphase contamination)

- ethanol ppt with 1 uL glyogen (glycoblue) and 1/10 volume 3M sodium acetate and 2.5X ethanol - overnight -80

- wash pellet with 80% ethanol and resuspend in 20 uL H2O

after the ethanol precipitation all my ChIP samples had a big white pellet, but my input looked like the normal tight blue pellet that I normally see. The pellet dissolved easily in the water. I don't think it is SDS because it didn't come out of solution when tubes were incubated on ice. I don't think it is salt because the input has the same amount of salt.

I nanodropped the samples and the A260/280 were ~0.6 for ChIPs and 1.8 for input. A260/230 were ~0.2 for ChIPs and ~2.2 for input. so I think its protein contamination.

I'm wondering if I should do another proteinase K digest and clean up again. Not sure if I should reverse the cross-links again with more salt? If anyone has had this problem and have found out how to fix it I would be so grateful!

I had planned on first performing qPCR then sequencing these samples, but I am sure that the protein is going to interfere with the downstream steps.