Pre-Sure ChIP Antibody Validation Kit

The Pre-Sure™ ChIP Antibody Validation Kit is a complete set of optimized reagents to determine the qualification of antibodies for use in chromatin immunoprecipitation (ChIP) using chromatin isolated from various species, particularly mammals, in a fast and high throughput format. This significantly reduces time and labor, as well as cost, versus having to conduct a full ChIP-PCR or ChIP-Seq experiment to validate antibody quality for ChIP-grade status. The kit has the following advantages:

• High Speed - The entire procedure can be completed in less than 3 hours with the actual handling time being less than 30 minutes.
• High Specificity - Detected signals are directly and specifically from the target protein-DNA complex captured by the antibody and fully consistent with results obtained by ChIP-qPCR and ChIP-seq. No need for PCR amplification and sequencing.
• High Sensitivity - As low as 0.5 ng of the enriched target protein-DNA complex can be detected.
• High Throughput - 96 strip-well plate format allow for validation of a single antibody each time or up to 43 antibodies simultaneously.
• High Convenience - An assay control (core chromatin), negative control non-immune IgG, and positive control antibody are provided in the kit, and are used for determination of ChIP enrichment efficiency, specificity of the validation test, whether test was successfully performed overall.

Background
The interaction between protein and DNA plays a critical role for cellular functions such as signal transduction, gene transcription, chromosome segregation, DNA replication and recombination, and epigenetic silencing. Identifying the genetic targets of DNA binding proteins and knowing the mechanisms of protein-DNA interactions are important for understanding cellular processes. Chromatin immunoprecipitation (ChIP) offers an advantageous tool for studying protein-DNA interactions, but requires high quality, ChIP-grade antibodies to achieve desirable results via PCR and sequencing/next-generation sequencing.

Principle & Procedure
This kit includes a positive control antibody, a negative control non-immune IgG, and core chromatin extract that can be used as a positive control to demonstrate the efficacy of the kit reagents and protocol. The antibodies are first bound to the assay wells. The positive control antibody AcH3 is ChIP-grade, which can immunoprecipitate the acetylated histone H3 complexed with DNA in core chromatin and generate a fluorescent signal (read by a fluorescence microplate reader at 490ex/530em) in the reaction with the Fluoro Assay Solution. Similarly, if the tested antibody works in ChIP, it can also capture target protein-DNA complex from chromatin and generate a fluorescent signal. The signal intensity is proportional to the amount of protein-DNA complexes captured by the antibody, and accordingly indicates ChIP-grade status as well as the grade intensity.

Starting Materials
Original source materials can include various tissue or cell samples such as cultured cells from a flask or dish. Chromatin can be isolated and sheared using Epigentek's ChromaFlash Chromatin Isolation & Shearing Kit or your own successful method. The amount of input chromatin can be 0.2 µg (about 2x10⁴ cells) to 5 µg (about 0.5 x 10⁶ cells) depending on whether or not the antibody-targeting proteins are highly abundant. The optimal reaction should be 1 µg (about 1x10⁵ cells) of input chromatin since enrichment of target proteins to genome loci varies and some target proteins are of low abundance. Each input antibody to be tested should be 0.6 µg per test well.

 
 Fig. 3. Comparative demonstration of the Pre-Sure ChIP Antibody Validation Kit's antibody validation data versus an actual ChIP-qPCR experiment with the same antibodies. PCR results of successful ChIP antibodies and unsuccessful ChIP antibodies were consistently matched to higher and lower relative fluorescent units generated by the kit within designated ranges, respectively. Top: qPCR amplification of GAPDH in euchromatin after ChIP with various antibodies. Bottom: Fluorescent signal intensity obtained using the Pre-Sure kit in comparison to ChIP-qPCR Ct values.

 Fig. 1. Schematic procedure of the Pre-Sure™ ChIP Antibody Validation Kit.

 Fig. 2. ChIP-grade Antibody Validation: Core chromatin isolated from Hela cells was used for measuring "ChIP-Grade Intensity" (CGI) of each antibody using the Pre-Sure ChIP Antibody Validation Kit. To confirm the CGI results, ChIP-qPCR analysis was performed using the same antibodies to enrich the protein or modified histone-DNA complexes on euchromatin. A euchromatic GAPDH locus served as a reference. The antibodies H3K4me2 (3), H3K9ac (4), DNMT1 (5), H3K18ac (6), and H3K14ac (7), which are demonstrated to be ChIP-grade by epigenetic researchers, highly enriched the  proteins/modified histones associated with euchromatin, while antibodies HDAC4 (1) and H3K9me2 (2) which are demonstrated to be less associated with euchromatin, show little to no enrichment, and considered non-ChIP-grade.