Ampification in no-antibody control of ChIP assay - High background - (Dec/27/2004 )
I'm using the Fast Chip method (using diagenode 1-day chip kit). I am having serious no ab problems! There's no preclear step for the binding beads. I tried more washes. Any ideas?
Okay... so I precleared samples (I will check to se if that has a difference). I was going to check to see if block the beads helped as well, but when I blocked the beads with BSA for an hour - after spinning there was barely any pellet for the beads? What happened to the beads? Can BSA chew them up or something? Hopefully the preclear step does the trick.
I have also amplification in a no-antibody control (IP-) of the ChIP assay. I'm using Fibroblast cellines and doing the precleaning for 1 hr and I use 120 ul protA/G beats. I's washing 5 times 5 min. with the different washbuffers in the Upstate kit. And my background is as high as my IP samples.
Can somebody help me??
Does ChIP actually ever work, do the no antibody samples ever give a low signal? Argg
You should use same amount of negative control IgG from same species, should include a positive control. The other way is to do PCR with a negative control gene promoter and to subtract from the negative control gene promoter, then calculate fold enrichment after subtracting from negative control promoter. I used a fast Chromatin IP kit from AmericanGeneTech.com and found it to be very simple and fast (4 hours) with much less nonspecific issues. It is in strip well format and can be done in high throughput.