Nonspecific precipitation in ChIP - (Jan/27/2006 )
one problem haunts me these days in my ChIP assay. Everything is OK in my ChIP: the sample with antibody has correct band, the negative sample has no band, BUT, one sample without antibody has band always!! I extended the wash time, all the band disappear, including the correct and the wrong band; I shortened wash time, all band appear again! Ohhh, , i have no idea how to modify it now. Could you give me some advice?
btw, i use the selfmade solution in ChIP.
THANKS A LOT!!
Do you block the beads with non-specific DNA (salmon sperm DNA) prior to running your IP.
O, shall I? I did not add any non-specific dna, but only protein A sepharose slurry 30mins, and then antibody overnight, and then protein A+G sepharose slurry. I noticed that these non-specific dna is not contained in many protocols. It is necessary? I will try it. Thank you.
Could you tell me the volume of salmon sperm dna, or give me one copy of your protocol? really appreciated.
Yes, standard protocol doesn't include the preclean step. If there is a problem, precleaning may help. Check this post for protocol
Thanks a lot! This really helps.
BTW, today is Chinese New Year, Happy new year to you guys.