Imunoprecipitação da Cromatina
What is Chromatin Immunoprecipitation?
Chromatin Immunoprecipitation is used to link specific states of chromatin to individual loci in a cell, to understand how genes are regulated, and to decipher theHistone Code. Chromatin IP was first described by the group of James Broach in a 1993 publication (Genes & Dev. 1993 7:592-604) that studied the association of histone acetylation state with transcriptional gene silencing in yeast. The technique was first used successfully on mammalian cells by Richard Treisman's group, published in 1998 (Cell (1998) 92:475-87). The basis for Chromatin Immunoprecipitation is to fix DNA to chromatin proteins by treating cells with formaldehyde, then extract the chromatin, shear it to workable fragment sizes, and enrich for chromatin fragments of interest by immunoprecipitation with antibodies specific for chromatin proteins, or modifications on those chromatin proteins. Initially, the retrieved chromatin was analyzed by immobilization on nitrocellulose and probing with labeled DNA complementary to the loci of interest. This approach was too constraining on the sensitivity of the technique, so soon PCR-based readouts were developed. At first these consisted of endpoint-PCR assays and gel electrophoresis to visualize the amplified fragments. One limitation of endpoint-PCR is that exponential amplification of target sequences tends to occur within a narrow range of PCR cycles, after which it becomes inefficient and is eventually exhausted. Endpoint PCR could not convey the information of which cycle numbers resulted in optimal amplification. A better method for quantifying locus-specific DNA recovered from Chromatin Immunoprecipitation involves real-time quantitative PCR, and determination of the number of cycles required for the signal to cross the threshold of background. This is referred to as the Ct value, and is expressed as the cycle number at which the threshold is crossed. The user must still validate that the correct sequence is being amplified, which usually involves gel electrophoresis to visualize the amplified fragment. For gene-specific chromatin Immunoprecipitation, real-time qPCR is probably the most widely used and reliable technique. Further developments of the technique involved probing microarrays with recovered DNAs, sometimes with Whole Genome Amplification to increase sensitivity. Finally, the newest and potentially most powerful detection technique for detecting ChIP DNA is the direct sequencing of recovered DNA, often called ChIP-Seq. An important consideration when using ChIP-Seq is to avoid older Chromatin IP Kits that use non-mammalian DNA to block the protein G beads, as these DNA sequences will contaminate the sequencing reactions. Active Motif's ChIP-IT® Express Kits are suitable for ChIP-Seq, enabling users to easily transition their ChIP reactions into Next Generation Sequencing platforms.
The central role of Chromatin IP in epigenetic studies
ChIP is the principal technique used to map epigenetic marks to individual loci in the genome. As epigenetic researchers determine the relationships between histone marks and gene expression, or between the binding of non-histone proteins, including transcription factors, to chromatin, and their effects on gene expression, it is necessary to immunoprecipitate chromatin and determine which loci are enriched by the specific immunoprecipitations. Chromatin Immunoprecipitation is also a very technically challenging method, and numerous factors can cause it to fail, so researchers who are not experts in the techniques are best served using well-validated and reliable kits to perform these assays.
Active Motif's innovation in developing our ChIP-IT® kits and optimized reagents and protocols make for the most sensitive ChIP kits available on the market. ChIP-IT Express Kits make chromatin immunoprecipitation (ChIP) faster and more consistent by providing you with all of the critical components needed in a single Chromatin Immunoprecipitation kit, validated and proven to work in ChIP. ChIP-IT® Expresschromatin immunoprecipitation kits are a marked improvement over traditional Chromatin IP because they utilize protein G-coated magnetic beads, making it possible to perform ChIP in just 1 day. However, for researchers looking to achieve the ultimate sensitivity for detection of low abundance proteins such as transcription factors, Active Motif's ChIP-IT® High Sensitivity Kit is the perfect solution. In addition to working for low abundance proteins, and low binding affinity ChIP antibodies, the ChIP-IT High Sensitivity Kit is also ideal for use with limited sample material as successful ChIP reactions have been performed with as few as 1,000 cells per immunoprecipitation reactions.
ChIP, ChIP-Seq and ChIP-chip Validated Antibodies
Over the last 4 years, Active Motif has developed the best-characterized lineup ofChromatin Immunoprecipitation-validated primary antibodies on the market, and is the New Leader in all chromatin immunoprecipitation applications. Our lineup is developed by us internally, or in collaboration with leading scientists in the field, to produce only top-quality antibodies. Check out our line of ChIP-validated antibodiesfor your experiments! In addition, Active Motif is now in the process of re-evaluating our large collections of histone modification and transcription factor antibodies for use inChIP-Seq and ChIP-chip.
Preparing Chromatin: Sonication or Enzymatic Shearing?
The first step in successful chromatin immunoprecipitation is shearing the chromatin into 200-1000 bp fragments. This has traditionally been performed by subjecting the isolated chromatin to different pulses of sonication. Sonication is an effective method for shearing DNA, and is the most widely-used method. To ensure you get the best sonication results possible, Active Motif offers a variety of EpiShear™ Sonicators & Accessories, including a Multi-Sample Sonicator that can shear up to 8 samples simultaneously and a Probe Sonicator that offers the flexibility to shear both small or large sample sizes.
In some circumstances, however, it can be difficult to optimize sonication shearing due to complications arising from emulsification and overheating of the sample. Because of this, or if you don't have a sonicator, Active Motif has developed a robust and user-friendly method to shear chromatin by enzymatic treatment. The ChIP-IT® Express Enzymatic Shearing Kit uses a proprietary Enzymatic Shearing Cocktail that quickly shears DNA into 200-1000 bp fragments. Because enzymatic shearing is solely time and temperature dependent, the problems associated with sonication are eliminated and ChIP results are improved.
Whether you choose to shear your chromatin by sonication or enzymatically, the first step in preparing your sample is cell lysis. To get the highest yields and ensure that the lysate is representative of the entire cell, it is critical to completely lyse your sample. This can be difficult when working with tissue, as well as cell lines that are resistant to lysis under normal detergent conditions. Active Motif's Dounce Homogenizers, which physically disrupt cell membranes, are the best way to prepare lysates that are of the highest quality, yield and concentration possible.
Key advantages of the Active Motif ChIP-IT® Express platform
The increase in efficiency of chromatin immunoprecipitation afforded by the inclusion of protein G-conjugated magnetic beads in ChIP-IT Express has made it possible to develop new kits that dramatically extend the utility of Chromatin Immunoprecipitation. These new kits include the first kit that enables the processing of 96 chromatin immunoprecipitation reactions simultaneously, ChIP-IT® Express HT, the first kit for performing sequential chromatin immunoprecipitation, Re-ChIP-IT®, the first kit for studying the involvement of non-coding RNA in epigenetic processes, RNA ChIP-IT®, the first kit for studying DNA:protein interactions from formalin-fixed, paraffin-embedded (FFPE) tissue samples, ChIP-IT® FFPE, as well as the first kit to enable chromatin extraction and ChIP to be performed from PBMCs, ChIP-IT® PBMC.
Whether you are studying histones, histone modifications, transcription factors or chromatin-associated proteins, ChIP-IT & ChIP-IT Express Chromatin Immunoprecipitation Kits will help you achieve your experimental goals.
ChIP-IT® Accessory Reagents
In addition to complete kits for magnetic ChIP, Active Motif offers species-specific ChIP-IT Control Kits and ChIP Control qPCR Primer Sets, a large number of chromatin immunoprecipitation (ChIP)-validated antibodies, Protein G Agarose Columns and Magnetic Beads, as well as Ready-to-ChIP Chromatin, all of which help you get better Chromatin IP results. To make data interpretation easier for the ChIP-IT® High Sensitivity Kit, we recommend the use of Active Motif's ChIP-IT® qPCR Analysis Kit.