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Specific but inefficient pull-down for ChIP - advice welcome!


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#1 Tom_Huxley_2001

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Posted 15 March 2011 - 07:36 PM

Hi,

Ok, I've got the Fast ChIP method up and running (Nelson et al., 2006). The method works nicely - however, I'm concerned about the low percent input values I'm getting. In papers and on this site I see people consistently report getting pol II pull-down at active genes (GAPDH promoter for example) that is 5-10% of input. I'm using the pol II ab from Santa Cruz Biotech that is commonly employed. I'm using monocytes for my ChIP assay. Here is a brief overview of my results:

~5,000,000 (per I.P.) cells lysed in standard 1% SDS buffer (0.2 ml) and sonicated on bioruptor to ~200-500 bp.

After pelleting debris, sample is diluted in 1:10 in Fast ChIP I.P. buffer (no SDS), 2 ug pol II ab or Rb IgG is used to IP o/n

Sample precipitated with Protein A/G agarose (Pierce) for 1 hr, washed 6X Fast ChiP I.P., incubated at 95 degrees for 10 min with 0.1 ml 10% Chelex-100, Proteinase K 55 degrees 30 min, then 95 degrees for 10 min. Collected super.

For SYBR-Green qPCR: input was 5 ul of sonicated chromatin mixed with 100 ul Chelex-100 and processed as above. The super was diluted 1:50 before qPCR.

RESULTS (standard GAPDH promoter primer set with efficiency of ~105%):

Rb IgG: undetectable signal (i.e. Ct >40)

Mean pol II: Ct values of 27.1, 27.0, 27.1

Mean input Ct value: 27.8

So although my pull-down is obviously specific, it is only ~0.1% of input. Any ideas why the pull-down is so low? Fixation? I do 1% formaldehyde for 15 min at RT. Antibody amount? I'm really at a loss as to how others are pulling down 50-fold more pol II-bound sequence than I am, and it makes be cautious to continue with the examination of factors I'm interested in until I improve these results.

Thanks in advance.

#2 KPDE

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Posted 17 March 2011 - 09:27 AM

Hi,

Ok, I've got the Fast ChIP method up and running (Nelson et al., 2006). The method works nicely - however, I'm concerned about the low percent input values I'm getting. In papers and on this site I see people consistently report getting pol II pull-down at active genes (GAPDH promoter for example) that is 5-10% of input. I'm using the pol II ab from Santa Cruz Biotech that is commonly employed. I'm using monocytes for my ChIP assay. Here is a brief overview of my results:

~5,000,000 (per I.P.) cells lysed in standard 1% SDS buffer (0.2 ml) and sonicated on bioruptor to ~200-500 bp.

After pelleting debris, sample is diluted in 1:10 in Fast ChIP I.P. buffer (no SDS), 2 ug pol II ab or Rb IgG is used to IP o/n

Sample precipitated with Protein A/G agarose (Pierce) for 1 hr, washed 6X Fast ChiP I.P., incubated at 95 degrees for 10 min with 0.1 ml 10% Chelex-100, Proteinase K 55 degrees 30 min, then 95 degrees for 10 min. Collected super.

For SYBR-Green qPCR: input was 5 ul of sonicated chromatin mixed with 100 ul Chelex-100 and processed as above. The super was diluted 1:50 before qPCR.

RESULTS (standard GAPDH promoter primer set with efficiency of ~105%):

Rb IgG: undetectable signal (i.e. Ct >40)

Mean pol II: Ct values of 27.1, 27.0, 27.1

Mean input Ct value: 27.8

So although my pull-down is obviously specific, it is only ~0.1% of input. Any ideas why the pull-down is so low? Fixation? I do 1% formaldehyde for 15 min at RT. Antibody amount? I'm really at a loss as to how others are pulling down 50-fold more pol II-bound sequence than I am, and it makes be cautious to continue with the examination of factors I'm interested in until I improve these results.

Thanks in advance.


Is the primer you are using, upstream of the transcription start site? You will probably get a lot more Pol II signal with a primer at, or a few hundred bp downstream of the TSS. Also, you are looking at the constitutively active GAPDH and not GAPDH-S, right? Only reason I ask is that I made the mistake of confusing the two as an early grad student. Also, are you using the N-terminal Pol II antibody or the CTD antibody (4H8). Not that the difference in binding between these two can explain your low binding but, once you get things working, to optimize your signal, the CTD antibody works better.




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