When researchers want to know what a protein is doing, they often take the direct route: Ask it directly. Is protein A hanging out with protein B? Try co-immunoprecipitation. Is protein A binding to nucleotide sequence B? Try ChIP.

ChIP, or chromatin immunoprecipitation, reports which DNA sequences a given protein binds to in the cell. The basic idea dates back decades, long before researchers could even fathom sequencing the human genome. Yet today,ChIP has become one of the foundational techniques of epigenomics, providing (when paired with next-generation DNA sequencing) a mechanism for locating and profiling genomic regulatory regions across the genome.

Researchers have tended to focus largely on protein-coding regions, if for no other reason than the ease of finding them, says Michael Snyder, professor and chair of genetics at the Stanford School of Medicine. “But pretty clearly, the regulatory regions are [also] important, and ChIP is one way and probably the best way of finding out where all these lie.”

In ChIP, protein-DNA interactions in cells are frozen by crosslinking, after which the cells are broken open and their chromatin released. That DNA is then fragmented into small pieces, and those carrying the protein of interest are pulled down using an antibody. Finally, the crosslinks are reversed and the recovered DNA is queried using either PCR, a DNA microarray or sequencing.

The basic idea—other than the readout methodologies—has remained largely unchanged for years. But that doesn’t mean tool developers have been idle. Here are some of the newest offerings.

Working with low cell numbers

ChIP traditionally has been performed using cultured cells, and lots of them. Some kits require inputs of a million cells or more. But for those working with clinical samples, populations purified via fluorescence-activated cell sorting (FACS) and formalin-fixed paraffin-embedded (FFPE) cells, for instance, such numbers can be tough to reach.

Active Motif’s ChIP-IT® High Sensitivity kit provides one option. The kit has a two-fold purpose, according to product literature: It can handle low cell numbers (as few as 1,000 cells) or low-abundance proteins (50,000 cells)—particularly the rare transcription factors that some researchers study via ChIP. “That’s currently one of our top-selling products, as people investigate ChIP at the transcription-factor level,” says Kyle Hondorp, the company’s product manager.

For those who would sequence the resulting DNA, Kapa Biosystems’ KAPA Hyper Prep Kit can build libraries from the low-DNA inputs such experiments produce, says Adriana Geldart, support and applications manager at Kapa. According to Geldart, the company has produced sequencing libraries from as little as 100 pg DNA. “A customer we are collaborating with consistently uses 10,000 cells,” she says.

And for those who prefer to outsource their epigenetics work, Active Motif recently launched its Low Cell Number ChIP-Seq service. It requires at least 10,000 cells, Hondorp says.

Solid-state ChIP

ChIP traditionally has been performed using agarose or magnetic beads coupled to protein A or protein G to precipitate antibody-chromatin complexes. One company, though, has transformed the process into a filter-based method.

The Chromatrap assay, commercialized by Chromatrap, uses a protein A- or protein G-coated porous filter as a capture reagent instead of beads, removing many of the traditional liquid-handling steps associated with ChIP.

“It’s basically a solid-state ChIP assay,” says Stephen Knight, director of sales and marketing at Chromatrap.

Among other things, says product development manager Amy Beynon, that format means users never need worry about losing their sample during washing and pipetting steps. “When we load a sample, it stays within the matrix,” she says. “And the washes are just 30 seconds instead of five-minute washes with magnetic beads.”

Indeed, the assay can shorten ChIP “dramatically”—from days, in some cases, to as little as five hours, says Knight.

Chromatrap assays are available in multiple formats, including single spin columns of 96-well microplates, with kits optimized for qPCR and/or sequencing as a downstream process. Sonication and enzymatic formats also are available. “We have 36 kits,” Knight says, expanding to “nearly 40” by the end of 2015 and to 50 by the end of the first quarter of 2016.

The company will be launching a native (i.e., no crosslinking) Chromatrap kit in January, Knight says, followed shortly thereafter by a kit for working with FFPE samples.

“Reverse ChIP”

ChIP enables researchers to identify genomic regions associated with a given protein. But what if they want to know what proteins (or RNAs, or DNA for that matter) are associated with a particular piece of genomic real estate? In 2013, Hodaka Fujii at Osaka University described a method for doing just that [1].

So-called enChIP (engineered DNA-binding molecule-mediated chromatin immunoprecipitation) uses a catalytically inactive CRISPR/Cas9 system and user-selected guide RNA to specifically capture the macromolecular neighbors of a selected piece of genomic DNA. Instead of using an antibody to a particular protein, the assay targets a peptide tag that is appended to the Cas9 protein.

In this way, researchers can identify whatever proteins, RNAs or other DNA segments are associated with the targeted DNA, says Hondorp. (Active Motif is commercializing the assay and plans to launch it in early 2016, she says.)

“Instead of coming at ChIP the traditional way, when you target a specific protein and identify where in the genome it is found, this is the reverse,” Hondorp explains: “Find a genomic region and identify what is bound there.”

Among other applications, Hondorp says, researchers can use the kit to measure chromatin looping (similar to Hi-C) or to identify off-targets of guide RNAs in CRISPR/Cas9 studies. “People who are interested in using a guide RNA for genome editing can use this kit to get specificity in their design,” she says.

Antibody considerations

ChIP assays are widely available commercially, but they are not strictly necessary. Paul Spear, product manager at Novus Biologicals, a Bio-Techne brand, says labs with extensive ChIP experience frequently use homemade reagents—like Snyder’s lab, for instance, which has ChIP’d several hundred antibodies as part of the ENCODE project. “There really hasn’t been a need to buy kits,” Snyder says.

In fact, there’s essentially only one reagent that’s crucial when it comes to ChIP, Snyder says: “It really comes down to how good the antibodies are.”

Not all antibodies function well in every assay, so vendors typically indicate whether their antibodies have been specifically validated for ChIP. Abcam has some 600 ChIP-validated antibodies available; Novus Biologicals, a Bio-Techne brand, offers about 400; and Chromatrap, which recently released its first batch of antibodies, has nine.

Some companies go to great lengths to ensure these antibodies will work as expected. For instance, Abcam assesses antibody specificity and cross-reactivity using, among other things, dot blots, ELISAs and Western blotting with different peptide competitors, says epigenetics specialist Davide Mantiero. Novus tests its histone-modification antibodies on peptide arrays.

But, says Snyder, users would be wise to validate their antibodies in-house, as well. In 2012, he and his colleagues in the ENCODE and modENCODE projects laid out a set of guidelines for antibodies used as part of the project [2]. Among other things, that report specifies two-step validation procedures for both antibodies targeting transcription factors and histone modifications, including such assays as peptide arrays, immunoprecipitation-mass spectrometry and RNAi.

“Only one in four or one in five” antibodies that his lab tests are suitable for doing ChIP work, Snyder says. (Chromatrap scientists report an antibody validation rate of 40% to 50% in their hands, Beynon says.)

At the very least, advises Mantiero, test your newly purchased antibodies using the company’s recommended protocol and controls. Abcam, he adds, conducts extensive testing so users don’t need to. “Ideally, they only need to do the minimal optimization before using [an antibody] in a test experiment,” he says.

References

[1] Fujita, T, Fujii, H, “Efficient isolation of specific genomic regions and identification of associated proteins by engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR,” Biochem Biophys Res Comm, 439, 132-6, 2013. [PMID: 23942116] 

[2] Landt, SG, et al., “ChIP-seq guidelines and practices of the ENCODE and modENCODE consortia,” Genome Res, 22:1813-31, 2012. [PMID: 22955991] 

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