The ChromaFlash™ High-Sensitivity ChIP Kit is a complete set of optimized reagents to carry out a successful chromatin immunoprecipitation procedure in a high throughput format starting from mammalian cells or tissues. The highly specific and sensitive kit is suitable for selective enrichment of a chromatin fraction containing specific DNA sequences using various mammalian cell/tissues. The optimized protocol and kit components reduce non-specific background ChIP levels to allow capture of low abundance protein/transcription factors and increased specific enrichment of target protein/DNA complexes. The target protein bound DNA prepared with the ChromaFlash™ High-Sensitivity ChIP Kit can be used for various downstream applications including PCR (ChIP-PCR), microarrays (ChIP-on-chip), and sequencing (ChIP-seq). The kit has the following advantages:
See also a quick chart to compare ChIP kits.
Background InformationProtein-DNA interaction 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 interaction is important for understanding cellular processes. Chromatin immunoprecipitation (ChIP) offers an advantageous tool for studying such protein-DNA interactions. It allows for the detection of a specific protein bound to a specific gene sequence in living cells using PCR (ChIP-PCR), microarrays (ChIP-chip), or sequencing (ChIP-seq). For example, measurement of the amount of methylated histone H3 at lysine 9 (meH3-K9) associated with a specific gene promoter region under various conditions can be achieved through a ChIP-PCR assay, while the recruitment of methylated H3-K9 to the promoters on a genome-wide scale can be detected by ChIP-on-chip or ChIP-sequencing. ChIP analysis requires that ChIPed DNA contains minimal background in order to reliably identify true TF-enriched regions. High background in ChIP is mainly caused ineffective wash buffers, insufficient cross-link reversal, inappropriate DNA fragment length, and residual RNA interference. To effectively capture TF/DNA complexes, which are often in low abundance, an ideal ChIP method requires having maximum sensitivity with minimized background levels. This method should also be able to enrich highly abundant protein/DNA complexes using a small amount of cells or tissues in a high throughput format. Epigentek’s ChromaFlash™ High-Sensitivity ChIP Kit is designed to achieve these goals by maximizing sensitivity and minimizing non-specific background signals.
Principle & ProcedureThis ChIP kit includes a positive control antibody (RNA polymerase II), a negative control non-immune IgG, and GAPDH primers that can be used as a positive control to demonstrate the efficacy of the kit reagents and protocol. RNA polymerase II is considered to be enriched in the GAPDH gene promoter that is expected to be undergoing transcription in most growing mammalian cells and can be immunoprecipitated by a RNA polymerase II antibody but not by non-immune IgG. Immunoprecipitated DNA is then cleaned, released, and eluted. Eluted DNA can be used for various downstream applications such as ChIP-PCR, ChIP-on-chip, and ChIP-seq.
Starting MaterialsStarting materials can include various tissue or cell samples such as cells from flask or plate cultured cells, fresh and frozen tissues, etc. In general, the amount of cells and tissues for each reaction can be 2 x 103 to 1 x 106 and 0.5 mg to 50 mg, respectively. For optimal preparation, the input amount should be 1 to 2 x 105 cells or 10 to 20 mg tissues since the enrichment of target proteins to genome loci may vary. For the target proteins that are low abundance transcription factors, the input amount should be 5 to 6 x 105 cells or 50 to 60 mg tissues.
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