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ChIP-IT® Express Kits make performing chromatin immunoprecipitation (ChIP, chromatin IP) more successful by combining all of the critical components needed in a single kit, including components for preparing chromatin using either sonication or enzymatic shearing. 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 Express Kits. For this reason, the ChIP-IT Express Shearing & ChIP-IT Express Enzymatic Shearing Kits are 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.
In addition, because Active Motif's EpiShear™ Sonicator was used to develop our ChIP-IT sonication products and protocols, it is ideal for use in preparing chromatin for use in ChIP.
| Name | Format | Cat No. | Price | |
|---|---|---|---|---|
| ChIP-IT® Express Shearing Kit (included in 53008) | 10 rxns | 53032 | $275 | Add to Cart |
| ChIP-IT® Express Enzymatic Shearing Kit (included in 53009) | 10 rxns | 53035 | $315 | Add to Cart |
Choose from sonication or enzymatic shearing
The first step in successful ChIP is shearing the chromatin into 200-1000 bp fragments. Active Motif offers two different methods for shearing chromatin. The more traditional method for shearing involves subjecting the isolated chromatin to repeated pulses of ultrasonic waves that are produced by a sonicator. The reagents included in the ChIP-IT Shearing Kit can be used with Active Motif's EpiShear Probe Sonicator to prepare your chromatin samples for use with our ChIP-IT Express Kits (Figure 1), as well as with other ChIP methods.
Although sonication is an effective method for shearing DNA, not everybody owns a sonicator or wishes to become proficient in its use. For new users, it can be difficult to optimize sonication parameters and to get reproducible results due to complications that can arise during sonication, such as emulsification and overheating of the sample. Because of this, Active Motif has developed a robust and extremely user-friendly method to shear chromatin for ChIP by digesting it. The ChIP-IT Enzymatic Shearing Kit uses a proprietary Enzymatic Shearing Cocktail that quickly shears DNA into 200-1000 bp fragments (Figure 2). Because enzymatic shearing is solely time and temperature dependent, the problems associated with sonication are eliminated. Enzymatic shearing is a good choice for users who do not do not plan to do enough ChIP to make it worthwhile to invest the time and money required to buy and learn how to use a sonicator.
Figure 1: Gel analysis of sheared chromatin prepared using the ChIP-IT Express Shearing Kit and the EpiShear Probe Sonicator.
HeLa cells were fixed for 10 minutes with 1% formaldehyde and then chromatin was prepared using the ChIP-IT Express Shearing Kit. Three samples of approximately 300 µl each were sheared with 5, 10 and 20 pulses at 25% amplitude using the EpiShear™ Probe Sonicator with a 1/8" microtip probe. Each pulse consisted of a 20-second sonication followed by a 30-second rest on ice to prevent heat build up. The sheared and unsheared chromatin samples were subjected to cross-link reversal, treated with Proteinase K and RNase A, then phenol/chloroform extracted and precipitated as described in the protocol. Samples were separated by electrophoresis through a 1% agarose gel. Optimally sheared chromatin will yield a smear between 200-1000 bp.
Lane 1: 100 to 1000 bp ladder.
Lane 2: HeLa DNA sheared for 5 pulses (optimal).
Lane 3: HeLa DNA sheared for 10 pulses (optimal).
Lane 4: HeLa DNA sheared for 20 pulses (over-sheared).
Note: From this experiment, the DNA sonicated for both 5 and 10 pulses are suitable for use in ChIP.
Figure 2: Gel analysis of of sheared chromatin prepared using the ChIP-IT Enzymatic Shearing Kit.
HeLa cells were fixed for 10 minutes with 1% formaldehyde and chromatin was prepared using the ChIP-IT 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: HeLa DNA incubated with the Enzymatic Shearing Cocktail for 5 minutes.
Lane 3: HeLa DNA incubated with the Enzymatic Shearing Cocktail for 10 minutes.
Lane 4: HeLa DNA incubated with the Enzymatic Shearing Cocktail for 15 minutes.
Flexible ordering options
The ChIP-IT Sonication and Enzymatic Shearing Kits provide sufficient shearing components to optimize shearing conditions and then make 5 preparations of sheared chromatin from three 15 cm plates (4.5 x 107 cells); each chromatin preparation will yield enough material to perform approximately 6 ChIP reactions. (These identical shearing components are already included in the ChIP-IT and ChIP-IT Express Kits, so you don’t need to buy a stand-alone shearing kit unless you’re running out of shearing components.)
Contents & Storage
Shearing Buffer (or Digestion Buffer and Enzymatic Shearing Cocktail), 10X PBS, 10X Glycine, 1X Lysis Buffer, Protease Inhibitor Cocktail, PMSF, EDTA, Proteinase K and RNase A. Reagent storage conditions vary from room temperature to -20°C. Please download a manual for complete kit specifications and storage conditions. All reagents are guaranteed stable for 6 months when stored properly.
