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ChIP-IT™ Kits make performing chromatin immunoprecipitation (ChIP) more successful by combining all of the critical components needed in a single kit, including shearing components. However, depending on how many immunoprecipitations you perform with each sample of sheared chromatin, you may run out of shearing components before using up all of the immunoprecipitation components in the ChIP-IT Kits. For this reason, the ChIP-IT Sonication & Enzymatic Shearing Kits are also sold separately. This enables you to buy only the reagents you need, while giving you the flexibility to use either traditional sonication or our unique enzymatic shearing.
Choose from sonication or reproducible enzymatic shearing
The first step in successful ChIP is shearing the chromatin into 200-1000 bp fragments. This has traditionally been performed by subjecting the isolated chromatin to different pulses of sonication. Although sonication is an effective method for shearing DNA, it can be difficult to optimize due to complications arising from emulsification and overheating. And, because the quality of your sheared sample depends greatly upon the quality of your sonicator, it may be necessary to purchase an expensive, "high-end" sonicator to get reproducible shearing. Because of this, Active Motif has developed a more robust and user-friendly method to shear chromatin for ChIP. The Enzymatic Shearing Kit uses a proprietary Enzymatic Shearing Cocktail that quickly shears DNA into 200-1000 bp fragments (Figure 1). Because enzymatic shearing is solely time and temperature dependent, the problems associated with sonication are eliminated and ChIP results are improved.
Figure 1: Analysis of DNA sheared using the Enzymatic Shearing Kit.
HeLa cells were fixed for 10 minutes with 1% formaldehyde and chromatin was prepared using the Enzymatic Shearing Kit. Chromatin was sheared with the Enzymatic Shearing Cocktail for 5, 10 & 15 minutes. The sheared and unsheared chromatin samples were subjected to cross-link reversal, treated with Proteinase K, phenol/chloroform extracted and precipitated as described in the protocol. Samples were separated by electrophoresis through a 1% agarose gel to assess shearing results:
Lane 1: 100 to 1000 bp ladder.
Lane 2: Unsheared HeLa DNA.
Lane 3: HeLa DNA treated for 5 minutes.
Lane 4: HeLa DNA treated for 10 minutes.
Lane 5: HeLa DNA treated for 15 minutes.
