ChIP PCR question - (Feb/22/2011 )

After calculating your %Inputs I got .019% for your His-tag Ab and .038% for your TF Ab. So if you subtract out your non-specific IP then your left with .019% Input, which is not great but the antibody to your TF may simply not be as good as some other antibodies, maybe check the literature for ChIPs done on the same TF and see what other groups got. It is good that you don't see any signal using the negative control primers, however I have found that when your specific IPs are this low that reproducibility becomes an issue. In my experiences it is best to always do a control ChIP just to convince your boss (and yourself) that you can successfully pull off the experiment. Maybe choose a TF that has a good antibody available and has already been published on so you can just use already validated primer sets. Perform the experiment, and compare your results with those that are published. I would also (if you already haven't) validate your TF antibody, some aren't suitable for ChIP, but showing that your Ab can detect your TF by western blot or IF may provide some valuable insight as to the efficacy of the antibody. Lastly, because your non-specific IP was half of you specific IP I believe, and literature supports that your data is not acceptable for claiming true enrichment of your TF at your site of interest. Maybe EMSA would be a technique worth investigating? Just a thought, anyways hope this helps!

Hi chabraha, thanks so much. Think I'll do a check of the ab to check their efficiency. Thanks so much again. By the way, can I just check a few more things. How much %input diff should I get to be a convincing enough number for enrichment? Is there a min number standard? Also, regarding the chip protocol, I preclear my sample using the relevant IgG isotype before incubating the actual conjugated ab with the precleared Sample. How important is it to preclear the sample? If I don't preclear, is it as good if I just do an additional ip using the isotype IgG for comparison? Thanks again

Ideally I like to see at least 100 fold enrichment for my specific antibody IP over my IgG (non-specific) IP at my positive control region. That is not a hard and fast rule, and I'm not sure there is one out there but you can imagine what you'd believe if you were reviewing a paper with ChIP data, so I'd go with whatever would convince you the data is significant. Also, I do not pre-clear my ChIPed material. I just dilute my sample 1:100 in ChIP dilution buffer, add my antibody, add my beads and let them go overnight. I always include an IgG IP for non-specific interactions. Just out of curiosity how many cell equivalents are you using per IP?

My protocols require me to dilute my sample 10x only. I use supposedly 25ug chromatin per ip. And if I back calculate that, it's about 1.25-1.5 million per ip. Is that too little/much? And I use 20ul beads for each ip.

100 fold over IgG? So my previous data is like only 2 fold right? Oh my god. 100 fold sounds impossible to attain. :x

Sorry I meant I dilute 1:10, just like you. I'd day your in the right ballpark for cell numbers I use ~2x10^6 cells per IP with 20ul Protein A magnetic beads. How about your elution method? 100 fold doesn't need to be reached by an means, remember, the antibodies your using are vastly different than the ones I'm using that's why its important to try and identify a positive control region where you know you TF of interest binds so you can truly be confident your IP and antibody are working effectively. Just a thought but you may want to try your IP with antibody alone overnight and then add in the beads for an hour or so or just add them together and let them go overnight,mix it up a bit and see if you can improve your IP efficiency. If you can't maybe try another antibody. Also, as long as your specific IP is pulling down your TF with statistical significance over your IgG IP you'd probably be ok.

Thanks for the info. I'm already doing the Ip overnight with the ab and beads and chromatin. But I'm not using magnetic beads though. I elute using pcr purification kit. Anyways, I'm already taking steps to check the ab binding efficiency using flow. And I've also searched papers to get some known positive ctrl primers. So i will check those out first brfore i continue trying. Thanks so much for helping me identify these steps. A question about checking the ab pulling efficiency. Do I need to check for those mock ctrls, like if I use anti his ab? That's because I don't have cells that express his tag. So do i use the same amount of ab as what I've determined for my ab of interest, or do I need to get some cells with his tag expression to determine empirically? Also, am I right to say that the amount of ab I use should not be saturated? I thought I read that somewhere before, though I'm not sure why. Or did I remember wrongly and they should be saturating?

If your using the anti-his as your "mock IP sample" you need to add the same amount of that antibody as your specific TF antibody for TF's I think 3ug is plenty. Also, I don't think you need to check the anti-his antibody for binding to your TF of interest. I would also just consider using whole IgG from rabbit as your "mock IP" instead of your anti-his and see if that drops your background

chabraha on Mon Feb 28 22:37:48 2011 said:

Hi, thanks for replying. I'm already in the midst of re-doing the IP, this time round using IgG isotype as the mock. I previously used anti-his Ab because that was what my colleague used. Wierd enough, when i did normal PCR, i keep getting bright bands from the anti-his samples, even brighter than my actual IP sample. I'll try to see if using the IgG isotype as mock will give cleaner result. Nevertheless, I'll just like to ask if you have any clue as to why such strong bands were seen with the anti-his samples? As a matter of fact, i even tried once using anti-flag, and too get such high background. Is that suggesting that somewhere in my protcol there might be a problem?

I have found that IgG gives me the lowest mock IP, once I used a GFP polyclonal and got a much higher mock IP. You may want to consider moving to magnetic beads......less background in my experiences. But, it simply may be that your antiboy towards your TF of interest is just not as good for ChIP, in that case you can switch antibodies or just do what you can with what you have......like subtract out your %Input for your your mock IP from your %Input of your specific IP. I use real time PCR on my samples so I never check bands, but I can tell you that my mock IPs always show up, but well after my specific IPs.

I would agree. Magnetic beads are definitely a great tool. You can always run an IP without anything to get an idea of what sort of background your beads may or may not be contributing.