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 Washing is easier because the spin steps have been replaced by rapid magnetic pull-down.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 2 in the Overview tab). 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 1 & 2). 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 1 & 2: 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.
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 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™ 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.