One of the times I performed this ChIP I got strong binding at the locus I amplified, but I have not been able to reproduce this result after many many tries. However, I did get the protein I am ChIPping to bind to the locus in one of the samples where it also bound the first time - so that is consistent and the antibody seems to be working? but it did not work for all of the ones it worked for the first time. I hope that makes sense. I am using the exact same tube of the exact same antibody. This is my protocol

Add 1% Formaldehyde directly to the media on my adherent cells for 10 minutes at RT

- The first time I may have added the formaldehyde to the cells a little warmer than the most recent attempt.

Quench with 0.125M glycine for 5 minutes at RT

- The first time I used glycine that came in a kit. I realize some of these things most likely don't matter but I am at a loss.

Wash 3x with cold PBS

Scrape and collect into PBS with inhibitors

- The first time I collected into 2 mL of PBS, but the most recent ChIP I collected into 5 mL of PBS, and after I spun down and resuspended in 1% SDS lysis buffer, my cell solution was viscous which it was not the first time around

Spin at 4 degrees for 5 minutes at 1000 rpm

Sonicate 15x 10 seconds on, 50 seconds off at 15 amps (got beautiful shearing)

- The most recent attempt I sonicated 20x - the shearing was good but not the EXACT SAME profile as the first time - still within 200 - 1000 bp though

Spin at 4 degrees for 10 minutes at 16,000 rcf and transfer to new tubes

Add dilution buffer

Add protein G agarose beads and incubate at 4 degrees for one hour on a rotator to preclear the sample

Spin at 4 degrees for 1 minute at 4800 rcf

Transfer supernatants to new tubes and remove 10% of the sample to use as input

Add antibodies and incubate at 4 degrees overnight on a rotator

Add protein G agarose beads and incubate at 4 degrees for 4 hours on a rotator

Spin at 4 degrees for 1 minute at 4800 rcf

Wash beads with low salt wash buffer, high salt wash buffer, LiCl wash buffer, and TE buffer (2 times)

Elute sample

Add 0.2M NaCl and incubate overnight in 65 degree water bath

- One note here - the most recent time I placed my samples in the bath when the temperature was at least 10 degrees, maybe 20 above 65 degrees for ~1 minute before I was able to correct my mistake

Add 1 uL of 10mg/mL RNase A and incubate for 30 minutes at 37 degrees in a dry bath

Add 40 ug/mL proteinase K and incubate for 2 hours in a 45 degree water bath

Perform a phenol/chloroform/isoamyl alcohol extraction

- the difference here is that the first time I used PIC at pH 5.2 (by mistake! I now realize this pH is for RNA extraction) which is why the most recent attempt I used PIC at pH 8.

- I performed the same amount of time for my incubation with 100% EtOH.

The reason I'm stumped is that even though these things were different between the two attempts, I still got the protein I am trying to IP to show up in one of my samples - just missing from one of the lanes where it showed up the first time.