Dear all,
I meet a big trouble in ChIP assay. I prepared the cell lysis buffer/shearing buffer containing 1%SDS which helps sonication well. However, 1%SDS damages the epitope of target protein and denature proteins, which prohibits the recognization of Antibody-target protein. Even though I diluted the sample 10-fold with 0.1% SDS before immunoprecipitation, the efficiency of ChIP is too low and I can only obtain 10 ng DNA sample (postive control: RNA Pol 2) from 25 ug chromatin materials per IP reaction. Then I reduced the concentration of SDS to 0.1% in the shearing buffer but the sonication efficiency is undesirable without high concentration SDS. The sonication analysis result is attached and turns out quite different efficiency with or without SDS.
By the way, I still have several other questions about ChIP assay.
1. Most of protocols suggest elution buffer containing 1%SDS and 0.1M NaHCO3. But the elution efficiency is not good enough. Can I replace it with 0.1 M Glycine-HCl pH=2.8 often used in Co-IP elution?
2. I usually use phenol/chloroform to purify DNA after reverse crosslink but I cannot see the DNA pellet on the 1.5 ml EP tube after ethanol precipitation and high-speed centrifuge. Generally speaking, how much DNA can be extracted from one ChIP reaction (2ug anti-RNA pol 2 antibody: 30 ul protein A/G beads: 25 ug sheared genomic DNA)
NO problem，just use 0.1%SDS+0.5%SDC，high fragment will not interfere with ChIp-seq results too much.
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