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Mock IgG versus No Antibody - Negative Control - (Dec/10/2009 )

I was just wondering what different people's thoughts were on using a mock antibody (e.g, Rabbit IgG) versus no antibody for their negative control in ChIP.

I'm currently working with a mock anitbody (Rabbit IgG from Cell Signaling) and am getting very high background (Ct's around 27-28, about the same as my experimental antibody). I've matched the antibody concentrations between the mock and my experimental antibody. I'm going to give the No antibody a shot tomorrow (incubating overnight tonight) and try decreasing my incubation time from overnight to 2 hours with the mock antibody and experimental antibody to see if this helps. But I've seen both mock and no antibody in the literature and was wondering what the folks in this forum thought about these options.

Thanks!

MM

-Mighty Mouse-

Mighty Mouse on Dec 10 2009, 02:45 PM said:

I was just wondering what different people's thoughts were on using a mock antibody (e.g, Rabbit IgG) versus no antibody for their negative control in ChIP.

I'm currently working with a mock anitbody (Rabbit IgG from Cell Signaling) and am getting very high background (Ct's around 27-28, about the same as my experimental antibody). I've matched the antibody concentrations between the mock and my experimental antibody. I'm going to give the No antibody a shot tomorrow (incubating overnight tonight) and try decreasing my incubation time from overnight to 2 hours with the mock antibody and experimental antibody to see if this helps. But I've seen both mock and no antibody in the literature and was wondering what the folks in this forum thought about these options.

Thanks!

MM


Using no antibody is a false control, and I think poor practice. If your IgG is giving you high background, switch IgGs or, better yet, use a non-specific antibody like anti-GFP, myc or FLAG (assuming you aren't expressing any of these tags in your system). I have been using anti-FLAG and typically have undetermined qRT-PCR signals, whereas the IgG I had used exhibited low background levels (CT 32-34).

-Dr Teeth-

I had a lot of trouble using IgG (very high background) in my Chips. This is how I solved it:

I changed my IgG for Cell Signalling IgG (very low background)
I changed my agarose+protein A/G beads for magnetic beads+ protein A (dynabeads)
Instead of "normal" pcr use Real Time PCR.

I hope it helps

-laurequillo-

laurequillo on Dec 11 2009, 09:50 AM said:

I had a lot of trouble using IgG (very high background) in my Chips. This is how I solved it:

I changed my IgG for Cell Signalling IgG (very low background)
I changed my agarose+protein A/G beads for magnetic beads+ protein A (dynabeads)
Instead of "normal" pcr use Real Time PCR.

I hope it helps


Hey...thank you both for your replies.

Dr. Teeth - I agree, it seems that a "No Antibody" control isn't really much of a control at all! If I continue to have problems with my mock IgG then I'll look into your suggestion. My only concern with your idea is price, wouldn't it be more expensive to obtain something like anti-GFP than Rabbit IgG? I haven't looked into it, but thats' my guess.

LQ - You make some great suggestions...unfortunately that's exactly what I'm doing! I have the magnetic beads from Millipore (Protein G, not Protein A...) and I'm getting very high background with the Rabbit IgG from Cell Signaling (Product 2729). Before running my current antibody (CREB, also from Cell Signaling) I ran a ChIP using the RNAPII antibody from Millipore and used their Mouse IgG as my mock antibody and had very little background. Finally, I'm already using qPCR.

I'm currently thinking that perhaps I got high background because I didn't spin down my samples after sonication and before removing my chromatin for the IP. If it works out I'll update everyone in case someone else runs into this issue.

Thanks

MM

-Mighty Mouse-

laurequillo on Dec 11 2009, 06:50 AM said:

I had a lot of trouble using IgG (very high background) in my Chips. This is how I solved it:

I changed my IgG for Cell Signalling IgG (very low background)
I changed my agarose+protein A/G beads for magnetic beads+ protein A (dynabeads)
Instead of "normal" pcr use Real Time PCR.

I hope it helps


Probably what made the greatest difference for you was the switch from agarose to magnetic beads. The non-specific IgG adds very little to background in our experience. We've seen no difference between using no antibody and IgG as mock when trying against several different antibodies and looking at several different genomic regions.

Even if you don't consider our empirical results, if you think about the number of potential differences between two sera (i.e. mock and specific antibody) then IgG isn't much of a control. Especially if you cast around for different IgGs till you find one that gives you the least background. The only reason to worry about using IgG as a control is if you think a reviewer will give you trouble over it when you submit the paper.

A better (but not perfect) control is to use your antibody of interest blocked with the peptide epitope used to make the antibody. The only problem with this is that the antibody's paratope could potentially be recognizing factors other than the epitope (though likely with a lower affinity) and thus blocking the paratope also could block non-specific binding in the mock but not the specific IP.

Another good control is to knock your factor of interest down with siRNA/shRNA and see if you get decreased enrichment in your ChIPed DNA. If your enrichment is decreased with siRNA at a particular region then the enrichment is specific to the factor of interest. This still has it's drawback with some factors if knocking them down drastically alters the architecture of the chromatin.

The truth is that there is no perfect control for background with ChIP. Thus it is always best to have a negative control region in the genome where it has been verified by other means that the protein of interest does not bind in your cell or tissue type and particular set of conditions. Whatever ChIP signal you get at this region can then be considered background. Of course this isn't always possible so you may have to just consider, after looking at several regions of the genome, that the region with the lowest ChIP signal represents your background.

-KPDE-

Mighty Mouse on Dec 11 2009, 04:25 PM said:

I'm currently thinking that perhaps I got high background because I didn't spin down my samples after sonication and before removing my chromatin for the IP. If it works out I'll update everyone in case someone else runs into this issue.

Thanks

MM



You donīt spin the samples down after sonication, maybe thatīs the reason.

-laurequillo-

Probably what made the greatest difference for you was the switch from agarose to magnetic beads. The non-specific IgG adds very little to background in our experience. We've seen no difference between using no antibody and IgG as mock when trying against several different antibodies and looking at several different genomic regions.
I second this - I find no meaningful difference looking at either higher-order repeat units or single-copy genomic loci...

-jhb80-