Need unusually stringent wash to reduce BG - (Feb/15/2010 )
Hi, I've cracked the ChIP protocol and things are working. For the most part. I want to thank all of you for your help. The chelex method has been a terrific timesaver when it comes to diagnostic ChIPs.
I'm scanning for a rather large array of Tx factors at the p21 locus. I keep getting BG... sometimes lots, sometimes very little.
I KNOW it's my washing steps, because if I increase the stringency, wash numbers, and wash times, my BG reduces significantly.
Problem is, I'm already using a high salt wash buffer with 750mM NaCl, 3 washes, 45 minutes each in addition to the standard low salt and LiCl washes. I'm using 500K cell equivalents per IP, generally 2ug of Ab (unless it's such a high affinity Ab that I need to use less), 10ul of Protein A dynabeads, 1% BSA, and .1ug/ul Salmon sperm DNA in a final volume of 500ul. I'm using home-brew reagents and dynabeads to save significant costs.
The RNA PolII positive control is working well, as are many of the Ab's I'm using. But there are some Ab's that just don't want to let go of the non-specific targets. I get no bands to speak of with beads-only control, so I question if pre-clearing the lysate would help.
My ultimate goal is ChIP-on-chip, so I need to get the BG as low as possible with maximal recovery of specific signal for total amplification.
Have any of you guys had to REALLY increase the stringency of the washes to get rid of the BG? I'm washing the bejeezus out of these beads, but so far TFIIH, CBP, TBP Ab's are being stubborn and giving high BG. However, using the same stringency, I'm almost washing all of my p300 signal away.
My question is this: Do you think I would be better off titrating the amount of lysate, increasing wash time and numbers, increasing NaCl concentration to 1M, or is there something else I'm totally missing? Has anyone ever been forced to use a high salt wash buffer with 1M NaCl? I've tried remaking my high salt wash buffer from the ground up 3 times to no avail. Maybe changing the formulation of my ChIP dilution buffer? I've tried RIPA buffer but it doesn't really give better results over the normal dilution buffer.
If your CTs aren't too high already I would suggest titrating your lysate first (going more dilute rather than more concentrated). I probably suggest this more than anything else only because it has been effective for me in increasing the difference between binding at the region of interest and the negative control region. However, I don't think I've ever been able to completely eliminate background binding except for those antibodies that don't seem to bind to anything. Then I get no background and no signal period.
MunkySpunk on Feb 15 2010, 09:07 AM said:
I'm scanning for a rather large array of Tx factors at the p21 locus. I keep getting BG... sometimes lots, sometimes very little.
I KNOW it's my washing steps, because if I increase the stringency, wash numbers, and wash times, my BG reduces significantly.
Problem is, I'm already using a high salt wash buffer with 750mM NaCl, 3 washes, 45 minutes each in addition to the standard low salt and LiCl washes. I'm using 500K cell equivalents per IP, generally 2ug of Ab (unless it's such a high affinity Ab that I need to use less), 10ul of Protein A dynabeads, 1% BSA, and .1ug/ul Salmon sperm DNA in a final volume of 500ul. I'm using home-brew reagents and dynabeads to save significant costs.
The RNA PolII positive control is working well, as are many of the Ab's I'm using. But there are some Ab's that just don't want to let go of the non-specific targets. I get no bands to speak of with beads-only control, so I question if pre-clearing the lysate would help.
My ultimate goal is ChIP-on-chip, so I need to get the BG as low as possible with maximal recovery of specific signal for total amplification.
Have any of you guys had to REALLY increase the stringency of the washes to get rid of the BG? I'm washing the bejeezus out of these beads, but so far TFIIH, CBP, TBP Ab's are being stubborn and giving high BG. However, using the same stringency, I'm almost washing all of my p300 signal away.
My question is this: Do you think I would be better off titrating the amount of lysate, increasing wash time and numbers, increasing NaCl concentration to 1M, or is there something else I'm totally missing? Has anyone ever been forced to use a high salt wash buffer with 1M NaCl? I've tried remaking my high salt wash buffer from the ground up 3 times to no avail. Maybe changing the formulation of my ChIP dilution buffer? I've tried RIPA buffer but it doesn't really give better results over the normal dilution buffer.