ChIP-IT^® Express Chromatin Immunoprecipitation Kits

ChIP-IT Express improves ChIP

ChIP-IT Express improves on traditional ChIP by reducing or eliminating several time-consuming steps. The kit's magnetic beads have much lower background than traditional agarose beads, so pre-clearing and blocking are no longer necessary. Washing is easier because the spin steps have been replaced by rapid magnetic pull-down. And, only 3 washes are required, as compared to 8 washes with traditional ChIP. This dramatically reduces the amount of hands-on time during the assay. This not only saves time, but it minimizes manipulations and eliminates the DNA loss that can occur during purification, helping to ensure sample-to-sample consistency.

Optimized buffers reduce background

While the magnetic beads and bar magnet included in ChIP-IT Express make ChIP much faster and easier, many of the kit's buffers have been optimized to provide you with better ChIP results. The Elution and Reverse Cross-linking Buffers help reduce the background, which enables you to discern effects that are smaller in magnitude. Reduced background also makes it possible to work with fewer cells (see Figure 2 in OVERVIEW).  

In addition to reduced background, ChIP-IT Express buffers eliminate the need for DNA purification, so you can simultaneously elute the DNA from the beads while reversing the cross-links. These simple protocol changes give you results quickly and easily while offering the freedom to simultaneously perform multiple ChIP assays: now you can even perform multiple ChIPs in PCR tubes with a multi-channel pipettor (Figures 1 & 2).

Powerful magnet

ChIP-IT Express Kits include a strong bar magnet that can be used to turn pipette tip boxes into magnetic stands for immunoprecipitation and washing. P1000 tip boxes can be used with Eppendorf tubes (Figure 3), while P200 boxes work with PCR strips (Figures 1 & 2). Commercially available magnetic tube stands, such as those from Ambion (Figure 4) and Promega, can also be used.

Figure 3: Use of a standard tip box for magnetic ChIP.

ChIP-IT Express Kits include a strong bar magnet that can be used to convert pipette tip boxes into a magnetic stand for washing the protein G-coated magnetic beads.

Figure 4: Use of an Ambion 6-tube Magnetic Stand with ChIP-IT Express.

Commercially available magnetic tube racks, such as those from Ambion and Promega, can be used with ChIP-IT Express.

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 using sonication. Although sonication is effective, many users find that complications caused by emulsification and overheating can lead to variable results. To improve reproducibility, Active Motif has developed a unique enzymatic shearing method. For your convenience, all ChIP-IT and ChIP-IT Enzymatic Kits are available in your choice of sonication or enzymatic shearing formats and stand-alone kits are also available. Click here to learn more about your options. And, to make you ChIP even simpler, we also offer Ready-to-ChIP Chromatin. Using our chromatin and ChIP-IT reagents, the only variable in your ChIP will be the antibody you are testing.

ChIP-IT Control Kits and ChIP-validated antibodies

The original ChIP-IT Kits contain human positive and negative controls; these simplify the task of troubleshooting your experiment, interpreting your results and validating antibodies for use in ChIP. However, once an antibody has been shown to work in ChIP, it may no longer be necessary to run the controls. Moreover, many of our customers have requested ChIP controls for species other than human. For these reasons, ChIP-IT Express Kits are provided without controls. By purchasing a species-specific ChIP-IT Control Kit separately you can get exactly what you want, without having to pay for controls that you may not need. In addition, Active Motif offers an ever-increasing number of ChIP-validated antibodies, which are ideal for use with all of our ChIP-IT Kits.

Chromatin immunoprecipitation (ChIP) involves the immunoprecipitation of protein/DNA complexes that have been stabilized via cross-linking. It offers a versatile solution by combining the specificity of immunoprecipitation, the sensitivity of PCR and the screening power of array profiling. However, ChIP can be technically challenging and difficult to validate without well-proven reagents. By providing proven antibodies, reagents and controls, ChIP-IT Kits guarantee the best results possible.

Figure 1: Schematic of chromatin immunoprecipitation using ChIP-IT Express.

In ChIP, intact cells are fixed using formaldehyde, which cross-links and therefore preserves protein/DNA interactions. DNA is then sheared into small uniform fragments and the DNA/protein complexes are immunoprecipitated using an antibody directed against the DNA-binding protein of interest. Following immunoprecipitation, the DNA is washed, cross-linking is reversed, and the proteins are removed by Proteinase K treatment. In the original ChIP-IT Kits, the DNA is then rapidly cleaned up using the included DNA purification columns. This DNA purification step in not necessary with ChIP-IT Express (Figure 1). The DNA is then screened to determine which genes were bound by the protein of interest.

Why use ChIP-IT^® Kits?

• Complete solution – all critical reagents needed are supplied
• Direct measurement of protein/DNA interactions, not just histone modifications
• Compatible with genome-wide profiling or selective PCR-based approaches
• No need to optimize reagents and protocol
• No phenol/chloroform extractions – DNA purification columns included in original ChIP-IT Kits, DNA purification step eliminated in ChIP-IT Express

Classically, ChIP has been performed with antibodies directed against abundant chromatin components, such as acetylated histones. While this yields information about transcriptional activity of promoters, it does not reveal which transcription factor is bound to the promoter(s) of interest. In contrast, ChIP using transcription factor-specific antibodies enables direct monitoring of transcription factor/DNA interactions. Unfortunately, this is technically more challenging than classical ChIP, and requires the preparation of several complicated buffers, inhibitor cocktails and blocking reagents. In addition, result validation is difficult without antibodies, controls and a protocol proven to work in ChIP. To overcome these problems and enable ChIP of both transcription factors and histones, Active Motif's ChIP-IT Kits offer a complete solution by providing nearly everything needed to successfully perform ChIP experiments, including positive and negative controls (Figure 2).

Figure 2: Improved chromatin immunoprecipitation using ChIP-IT Express.

A PCR analysis of immunoprecipitated DNA is shown. This figure was selected to demonstrate the efficiency of the ChIP-IT Express Kit. Typically ChIP requires 2 million cells per reaction. However, we have been able to reduce the amount of starting material and observed positive ChIP data from 100,000 cells or less.

HeLa cells were fixed for 10 minutes with 1% formaldehyde and then chromatin was prepared by sonication shearing (5 pulses). ChIP was performed in duplicate on chromatin isolated from 100,000 and 750,000 cells using a Negative Control IgG and an RNA pol II antibody. The DNA isolated through these ChIP reactions was then analyzed by 36 cycles of PCR using GAPDH positive control primers. (These antibodies and primers are available as the ChIP-IT Control Kit – Human. Kits for mouse and rat are also available.) Ten µl of each PCR was separated on a 1% agarose gel and visualized by UV-illumination following ethidium bromide staining. PCR using the GAPDH primers on DNA isolated with the RNA pol II antibody reproducibly generated more product than similar reactions performed on DNA isolated using the Negative Control IgG.These results demonstrate that ChIP performed with RNA pol II antibody greatly enriched for GAPDH promoter DNA, while ChIP performed with negative IgG did not.

Flexible ordering options

The ChIP-IT Express and ChIP-IT Express Enzymatic Kits include Protein G Magnetic Beads and the shearing components found in the ChIP-IT Shearing or Enzymatic Shearing Kits, respectively. However, depending on how many ChIPs 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 Sonication & Enzymatic Shearing Kits and Protein G Magnetic Beads are also sold separately.

ChIP-IT Express Kits provide sufficient components to perform 25 ChIP reactions. Sufficient shearing components are included to optimize shearing conditions and then make 5 preparations of sheared chromatin from three 15 cm plates (4.5 x 10⁷ cells); each of these chromatin preparations yield enough material to perform approximately 16 ChIP reactions. We recommend that you also purchase whichever ChIP-IT Control Kit is appropriate for your sample type, as using proven controls helps in troubleshooting, interpreting your results and validating antibodies for use in ChIP.

Contents & Storage

Protein G Magnetic Beads, Bar Magnet, Sticky Dots, Shearing Buffer (or Digestion Buffer and Enzymatic Shearing Cocktail), ChIP Buffers 1 and 2, Elution Buffer, Reverse Cross-linking Buffer, Wash and 10X PCR Buffers, 10X PBS, 10X Glycine, 1X Lysis Buffer, Protease Inhibitor Cocktail, PMSF, EDTA, Proteinase K, Proteinase K Stop Solution 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.