ChIP-IT® Express Chromatin Immunoprecipitation Kits

ChIP-IT-Express-Chromatin-Immunoprecipitation-Kits-AM16

Synonyms

Siliconized Tubes, 1.7 ml, eppendorf, chromatin assembly, chromatin immunoprecipitation, chip, sample preparations, vials

Name	Format	Cat No.	Price

Overview

ChIP-IT^® Express Overview

ChIP-IT Express Kit

ChIP-IT Express Enzymatic Kit

ChIP-IT Express Shearing Kit

ChIP-IT Express Enzymatic Shearing Kit

Siliconized Tubes

Chromatin Immunoprecipitation (ChIP) can be technically challenging and yield results that are difficult to interpret. Active Motif's ChIP-IT^® Express Kits eliminate some of these challenges by providing a streamlined protocol using protein G-coated magnetic beads, making it possible to perform ChIP in just 1 day. Kits that use either sonication or enzymatic digestion for preparing sheared chromatin are available. Complete your experimental design with Active Motif's ChIP-IT^® Control Kits and qPCR Primer Sets to validate your results at every step. To see how these features compare to the other ChIP Kits we offer, see our ChIP Kit Selection Guide.

ChIP-IT^® Express Kit Highlights:

ChIP-IT Express sonication is ideal for high quality chromatin fragmentation of difficult-to-lyse cells, cultured cells and tissue samples
No sonicator, simply use our ChIP-IT Express Enzymatic kit for cultured cell lines
Fast protocol makes it possible to perform ChIP in just 1 day
Protein G magnetic beads provide low background enrichment and streamline wash steps

Looking for experts to perform the assay? Our Epigenetic Services team can perform ChIP, ChIP-Seq and many other genome-wide data generation and analysis services for you.

How does it work?

ChIP involves the immunoprecipitation of protein/DNA complexes that have been stabilized via cross-linking. First, 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. Eluted DNA can be purified with Active Motif's Chromatin IP DNA Purification Kit and is ready for downstream analysis via qPCR or Next-generation sequencing.

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

Method

ChIP-IT^® Express Method

ChIP-IT Express improves ChIP

ChIP-IT Express improves on traditional ChIP by reducing or eliminating several time-consuming steps. Because the supplied magnetic beads have much lower background than traditional agarose beads, pre-clearing and blocking steps 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 protocols. This dramatically reduces the amount of hands-on time during the assay. This not only saves time, but reducing the number of times the sample is manipulated minimizes DNA losses that can occur during wash steps, which helps improve yield as well as 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, 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 6 in the Data section). In addition, these buffers were formulated to work together, so you can now elute the DNA from the beads while simultaneously reversing the cross-links.

These simple improvements give you results quickly and easily while making it possible to perform many ChIP assays at the same time. With ChIP-IT Express you can perform multiple ChIPs in PCR tubes with a multi-channel pipettor (Figures 2 & 3). The method has even made it possible for us to develop ChIP-IT Express HT, a high-throughput ChIP kit that can process up to 96 samples together in a 96-well plate.

Figures 2 & 3: Multiple-sample ChIP using ChIP-IT Express.
Immunoprecipitation and wash steps are fast and easy with magnetic beads because the pellet forms on the side of the tube, even pulling completely out of the buffer. Because it is simple to remove the buffer without disturbing the pellet, it is now possible to perform multiple ChIPs in PCR strips using a multi-channel pipettor.

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 4), while P200 boxes work with PCR strips (Figures 2 & 3). Commercially available magnetic tube stands, such as those from Ambion (Figure 5) and Promega, can also be used.

Figure 4: 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 5: 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 also 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 is most commonly achieved by performing sonication with equipment like Active Motif's EpiShear™ or PIXUL™ Sonicators. However, sonication can be difficult to optimize due to complications that can be caused by emulsification and overheating. Because of this, or if you don't have a sonicator, Active Motif has developed a robust, user-friendly method to shear chromatin by enzymatic digestion. Because Active Motif offers both ChIP-IT Express and ChIP-IT Express Enzymatic Kits, you can choose whichever shearing method you prefer. For more complete information on these shearing methods, please click here to learn more about your options.

ChIP Accessory Products and ChIP & ChIP-Seq validated antibodies

To simplify the task of troubleshooting your experiment, interpreting your results and validating antibodies for use in ChIP, Active Motif offers species-specific (human, mouse and rat) ChIP-IT Control qPCR Kits that contain a positive control antibody, a bridging antibody to enhance binding affinity of mouse monoclonal antibodies, a negative control antibody to evaluate non-specific binding, and species-specific positive and negative control qPCR primers for use in real-time or endpoint PCR. We also offer over 35 ChIP Control qPCR Primer Sets for many common ChIP targets in human, mouse, rat, Zebrafish, Drosophila and yeast, as well as Ready-to-ChIP Chromatin from a number of ENCODE cell lines that have been optimally sheared by sonication and ChIP-validated. Finally, Active Motif offers an ever-increasing number of ChIP-validated and ChIP-Seq validated antibodies, which are ideal for use with all of our ChIP-IT Kits and ChIP Accessory Products.

ChIP-IT^® Express Contents & Storage

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 6 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.

ChIP-IT^® Express Contents & Storage

Flexible ordering options

Contents & Storage

Data

ChIP-IT^® Express Data

ChIP-IT Express provides high quality ChIP-seq data.

Figure 6: Histone H3K9ac ChIP-Seq data following immunoprecipitation using the ChIP-IT Express Kit.
Active Motif's ChIP-IT Express Kit was used to immunoprecipitate 25 µg chromatin from K-562 cells using 3 µg Active Motif's AbFlex^® Histone H3K9ac recombinant antibody (Catalog No. 91103). Following ChIP, Illumina-compatible sequencing libraries were prepared using the Next Gen DNA Library Kit and Next Gen Indexing Kits (Catalog Nos. 53216 and 53264) and sequenced using the NextSeq 500. Results show high quality ChIP-seq peaks across a region of chromosome 1.

ChIP qPCR data for Histone H3K9ac enrichment using ChIP-IT Express.

Figure 7: Histone H3K9ac ChIP-qPCR data following immunoprecipitation using the ChIP-IT Express Kit.
Active Motif's ChIP-IT Express Kit was used to immunoprecipitate 25 µg chromatin from K-562 cells using 3 µg Active Motif's AbFlex^® Histone H3K9ac recombinant antibody (Catalog No. 91103). Following ChIP, DNA was diluted 1:20 and qPCR was performed using Active Motif's Human Positive Control primer sets ACTB-1 and GAPDH-1 (Catalog Nos. 71003 and 71004) and Human Negative Control Primer Set 1 (Catalog No. 71001). Results are displayed as binding events detected per 1,000 cells and show enrichment for the positive control PCR primer sets and little to no enrichment for the negative control primer set.