Jeffrey Perkel has been a scientific writer and editor since 2000. He holds a PhD in Cell and Molecular Biology from the University of Pennsylvania, and did postdoctoral work at the University of Pennsylvania and at Harvard Medical School.
Control of gene expression fundamentally occurs at the level of protein-DNA interaction. Those interactions can be teased apart easily enough biochemically, for instance, using gel-shift assays. But if researchers want to know whether a particular piece of genomic DNA is actually occupied in the cell, they usually turn to chromatin immunoprecipitation (ChIP).
ChIP is an immunoprecipitation process to identify specific stretches of chromosomal DNA associated with a protein of interest. Cellular protein-DNA interactions are first locked in place with a crosslinking step. The chromatin is then isolated, sheared and immunoprecipitated with antibodies specific to the protein of interest. The crosslinking is reversed to release the precipitated DNA, which is then queried (relative to a control sample) using either quantitative PCR (qPCR), microarrays or DNA sequencing.
By far the most common detection method is qPCR, says Rizwan Farooqui, market segment manager for Protein Function Products at Thermo Scientific-Pierce, as it can be used both as a readout assay and as a way to quality-control ChIP’d DNA prior to sequencing or array analysis. “Probably 75% of the market use[s] qPCR, because everyone has access to [q]PCR machines,” Farooqui says. He estimates that a small but growing fraction—perhaps 10% to 15% of researchers—uses sequencing (a process called ChIP-Seq), and the rest use microarrays (a combination called ChIP-on-chip).
The only difference between these approaches is in the amount of data they produce and whether the approach is targeted (qPCR and ChIP-on-chip) or not (ChIP-Seq). But the ChIP method itself is more or less the same in each case, and the same as it was a decade ago. That doesn’t mean tool developers aren’t working to make the process easier and more efficient, however. New kits are making the process of ChIP more easily automated and more sensitive, and new antibodies are expanding the roster of ChIP-able protein targets.
ChIP-ready antibodies
By all accounts, the key to a successful ChIP experiment is the antibody used in the immunoprecipitation. The problem is that not all antibodies are “ChIP-able,” and testing different antibody preparations can be expensive.
“Not all antibodies work in ChIP,” says Karin Abarca Heidemann, director of R&D at Rockland Immunochemicals, an antibody developer and vendor. “It all depends on which epitope the antibody recognizes.”
Some antibodies recognize their antigen in a three-dimensional context, for instance. But others only recognize denatured protein or have an epitope that is buried in the intact molecule. Such an antibody would probably work well in a Western blot (which measures denatured proteins)—but not in ChIP.
A number of companies now offer ChIP-validated antibodies, antibodies that have been tested either in-house or at least in the literature. Rockland tests its forthcoming EpiPlus® primary antibodies, which recognize specific epigenetic methylation, acetylation and phosphorylation events, in-house, for instance. So does Thermo, which has some 100 ChIP-validated antibodies in its portfolio, according to Farooqui, and Active Motif, which has 162.
EMD Millipore lists nearly 90 ChIPAb+™ antibodies on its website. ChIPAb+ antibodies “are individually validated for chromatin precipitation, every lot, every time,” according to the company website. The antibodies are validated against known positive and negative loci and are shipped with a matched IgG negative control as well as PCR primers for the positive locus, says Michael Sturges, senior product manager for Epigenetics at EMD Millipore. “ChIPAb+ antibody primer sets are great for people who are new to ChIP or need a validated positive control for their ChIP experiments.”
Antibody validation
Those researchers working with newly discovered proteins or newly prepared antibodies must first validate their antibodies before using them in ChIP. Unfortunately, the only way to validate an antibody in ChIP is to use it in ChIP. “You do the assay,” says Heidemann.
Active Motif does offer a ChIP Antibody Validation Service if you wish to outsource that work. Or, you can do the validation yourself.
EMD Millipore’s histone peptide array, called AbSurance™, can help researchers gauge an antibody’s specificity. AbSurance is a pair of PVDF membranes; the first contains 46 modified forms of the histone H3 leader peptide spotted at two concentrations, and the second contains 44 forms of histones H2A, H2B and H4.
“These are the same peptide macro arrays we use in our own histone antibody development,” Sturges explains. Using AbSurance, a researcher could determine, for instance, whether an antibody that recognizes histone H3 trimethylated on lysine-4 also recognizes that same modification in another location on another histone, or the mono- or di-methylated forms of that same amino acid.
Knowing an antibody’s specificity (or lack thereof) enables researchers to make more-informed choices in experimental design. “You can ask, is it an antibody I want to use or not?” Sturges says.
ChIP kits
New ChIP kits are also available. Earlier this year, Thermo Fisher Scientific launched a Magnetic ChIP Kit that substitutes the usual agarose beads of immunoprecipitation for paramagnetic beads. According to Farooqui, magnetic beads simplify ChIP processing, especially in high-throughput settings that use automation, as they obviate centrifugation steps.
Magnetic ChIP kits are also more sensitive, Sturges adds, noting that in one case agarose beads produced a 40- to 50-fold sequence enrichment over background, compared with 800-fold with magnetic particles. “That increase in fold enrichment delivers higher sensitivity,” he says.
Active Motif recently launched its own high-sensitivity product, the ChIP-IT® High Sensitivity Kit. According to Kyle Hondorp, product manager at Active Motif, this high-sensitivity product differs from standard ChIP kits in that it reduces the background signal and optimizes enrichment.
Hondorp says researchers using the High Sensitivity Kit can detect low-abundance transcription factors that are below the detection limits of traditional ChIP methods. It’s sensitive to proteins that are present at just 0.025% of the input sample, vs. other kits that require at least 1% abundance of the input. “It’s a completely different scale,” she says.
Furthermore, says Hondorp, the kit can use less starting material—as little as 1,000 cell equivalents—and still obtain specific enrichment. That’s just 1% of the 100,000 cell equivalents required by Active Motif’s other ChIP kits.
(Active Motif has bundled its High Sensitivity Kit in another product, the ChIP-IT ChIP-Seq kit, for those who intend to query their ChIP’d DNA using next-gen sequencing. That kit also includes the ChIP-IT qPCR Analysis Kit, which provides a way of quality-controlling ChIP’d DNA prior to sequencing.)
EMD Millipore’s high-sensitivity offering is the soon-to-be-launched Magna ChIP™ HiSens kit, which can be used for as few as 10,000 input genomes, says Sturges. (The typical ChIP input is closer to one million input genomes.) According to Sturges, the HiSens kit also uses paramagnetic beads, which are coupled to both proteins A and G to enable a wider range of precipitatable antibodies.
“Using the A/G blends in conjunction with the HiSens buffers, we get really stellar signal-to-noise ratios,” Sturges says. “If you are working with low amounts of cells, you’re likely to recover low amounts of material. Of course, the lower you can make your background noise, the more it’s going to make your signal pop.”
Also from EMD Millipore is the company’s forthcoming PureEpi™ Chromatin Preparation and Optimization Kit, which produces high-quality chromatin for use in ChIP. According to Sturges, the kit reflects the company’s R&D experience in producing the best-quality chromatin for the best possible ChIP results. “What we are releasing is a kit that includes a set of reagents as well as a protocol with detailed optimization guidelines and examples of data,” he says. “In short, we developed a kit to enable labs to produce chromatin that performs well in ChIP.”
Of course, these products represent merely a slice of the ChIP market; several vendors offer ChIP-ready antibodies and kits, and in many cases any one of them will serve you well. But for those who could use some technical help, EMD Millipore has produced a technical manual, the Guide to Chromatin Immunoprecipitation: Critical Factors for Success. By all accounts, ChIP is simple in concept but tricky in practice; this 32-page guide could be a good place to start ironing out the kinks.