Dear colleagues,
I am doing ChIP experiments on material from cell lines using Mandrup's protocol (Article Genome-Wide Profiling of Peroxisome Proliferator-Activated R...
) with some changes. I sonicated the samples, as usually, on Bioraptor 2x 5min (30sec on, 30sec off) and got fragments ~200bp (PIC1). As fragments were too short I shortened the sonication time to 2x 3min (30sec on, 30sec off) and fragments were now 500bp but a huge fraction of material was not sonicated (PIC2). I checked both batches of samples using qPCR, and CT values looked completely fine, but on Agilent, I got really confusing results... The first batch of samples (this with fragments ~200bp) did not show any material (PIC3; samples 4-11) and the second (fragments ~500bp and not sonicated fraction) exhibited only the long fragments (average size is more than 4000bp! (PIC4; samples 3-10). What could happen? I can imagine that too short fragments cannot be fished out from the solution by the antibodies, but what about the second situation? How could antibodies bind only to not sonicated fragments, ignoring these ~500bp? I was planning to do sequencing on these ChIP samples but now I'm stuck and completely have no idea what to do next.More Hanna Nowicka's questions See All
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