The ChromaFlash High Sensitivity Chip Kit is a complete set of reagents optimized to successfully perform a chromatin immunoprecipitation procedure in a high throughput format from mammalian cells or tissues. The highly specific and sensitive kit is suitable for the selective enrichment of a chromatin fraction containing specific DNA sequences using various mammalian cells/tissues. The optimized protocol and kit components reduce non-specific background ChIP levels to allow capture of low abundance protein/transcription factors and higher specific enrichment of target DNA / protein complexes.
Target protein-bound DNA prepared with the high-sensitivity ChIP ChromaFlash ™ 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:
The highly efficient enrichment ratio of the positive to negative control is> 500. An extremely low number of cells (as low as 2,000 cells per ChIP reaction) can be used to enrich highly abundant protein / DNA complexes.
Optimized buffers and protocol allow for minimal ChIP background by overcoming weaknesses that cause non-specific enrichment, thereby increasing the sensitivity and specificity of the ChIP reaction.
Increased antibody selectivity and capture efficiency through the use of unique chimeric proteins that contain the maximum number of IgG binding domains coated in the strip wells. This allows a strong binding of any subtype of IgG antibodies within a wide pH range, regardless of whether they are in monoclonal or polyclonal form.
5-hour rapid procedure, from input chromatin to ready-to-use DNA eluate.
The 96-well plate format makes the assay flexible. Or (a) manual with only one reaction at a time; or (b) high yield with 24-48 reactions each time.
Extensive top-down analysis support. Supports various downstream analysis workflows, including ChIP-PCR, ChIP-on-chip, and ChIP-seq.
Protein-DNA interaction plays a fundamental role in 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 understanding the mechanisms of protein-DNA interaction is important to understanding cellular processes. Chromatin immunoprecipitation (ChIP) offers an advantageous tool for studying such protein-DNA interactions.
It allows the detection of a specific protein bound to a specific genetic sequence in living cells by means of PCR (ChIP-PCR), microarrays (ChIP-chip), or sequencing (ChIP-seq). For example, measurement of the amount of lysine-methylated histone H3 (meH3-K9) associated with a promoter region of a specific gene under various conditions can be achieved by a ChIP-PCR assay, while recruitment of H3- K9 methylated promoters on a genome-wide scale can be detected by ChIP-on-chip or ChIP-sequencing.
ChIP analysis requires that the ChIPed DNA contain a minimum of background to reliably identify true TF-enriched regions. The high background level in ChIP is primarily due to ineffective wash buffers, insufficient cross-link inversion, 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 maximum sensitivity with minimized background levels.
This method should also be able to enrich very abundant protein / DNA complexes using a small number of cells or tissues in a high throughput format. Epigentek’s ChromaFlash ™ High Sensitivity Chip Kit is designed to achieve these objectives by maximizing sensitivity and minimizing non-specific background signals.
Principle and procedure
This 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’s reagents and protocol. RNA polymerase II is considered to be enriched in the GAPDH gene promoter which is expected to be transcribed in most growing mammalian cells and which can be immunoprecipitated by an RNA polymerase II antibody but not by non-immune IgG. Then the immunoprecipitated DNA was cleaned