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ChromaFlash High-Sensitivity ChIP Kit

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For immunoprecipitation of chromatin (ChIP) from small amounts of mammalian cells or tissues

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Suggested Workflow
Chromatin Isolation
 
 
Chromatin Shearing
 
 
ChIP
 
 
NGS Analysis
 
Schematic procedure for using the ChromaFlash High-Sensitivity ChiP Kit. (Cat. No. P-2027)
Low abundance protein enrichment: Sheared chromatin isolated from different numbers of MCF-7 cells was used for ChIP-qPCR analysis of ER-a enrichment in TFF1 promoters using the ChromaFlash High-Sensitivity ChIP Kit (Cat. No. P-2027) and the EpiQuik Quantitative PCR Fast Kit (Cat. No. P-1029).
High abundance protein enrichment: Sheared chromatin isolated from different numbers of MBD-231 cells was used for ChIP-qPCR analysis of RNA polymerase II enrichment in GAPDH promoters using the ChromaFlash High-Sensitivity ChIP Kit (Cat. No. P-2027) and the EpiQuik Quantitative PCR Fast Kit (Cat. No. P-1029).
Histone H3K18ac ChIP assay was carried out using the ChromaFlash High-Sensitivity ChIP Kit (Cat. No. P-2027). ChIP-Seq reads align with the same peak sites as ENCODE data.
Input Type: Chromatin
Research Area: Chromatin & Transcription
Target Application: Immunoprecipitation
Vessel Format: 96-Well Plate
100% Guarantee: 6 months
Catalog No.SizePriceQty
P-2027-2424 reactions $262.00 
P-2027-4848 reactions $399.00 
Availability: Usually Ships In 1-2 Days 
Product Overview

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:

  • The highly efficient enrichment ratio of positive to negative control is > 500. An extremely low number of cells (as low as 2,000 cells per ChIP reaction) can be used for enriching highly abundant protein/DNA complexes.
  • Optimized buffers and protocol allow minimal ChIP background by overcoming the weaknesses that cause non-specific enrichment, thereby increasing sensitivity and specificity of the ChIP reaction.
  • Increased antibody selectivity and capture efficiency through the use of unique chimeric proteins containing the maximum number of IgG binding domains coated on the strip-wells. This allows strong binding of any IgG subtype antibodies within a wide pH range regardless if they are in monoclonal or polyclonal form. 
  • Fast 5 hour procedure, from input chromatin to ready-to-use DNA eluate.
  • 96-well plate format makes the assay flexible. Either (a) manual with one single reaction each time; or (b) high throughput with 24-48 reactions each time.
  • Wide downstream analysis compatibility. Compatible with various downstream analysis workflows including ChIP-PCR, ChIP-on-chip, and ChIP-seq.

See also a quick chart to compare ChIP kits.

Background Information
Protein-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 & 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 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 Materials
Starting 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.

Product Components

WB (Wash Buffer)
AB (Antibody Buffer)
LB (Lysis Buffer)
CB (ChIP Buffer)
DRB (DNA Release Buffer)
DBS (DNA Binding Solution)
BS (Blocker Solution)
DEB (DNA Elution Buffer)
Enrichment Enhancer*
Protease Inhibitor Cocktail (PIC)*
Non-Immune IgG (1 mg/ml)*
Anti-RNA Polymerase II (1 mg/ml)*
Proteinase K (10 mg/ml)*
RNase A (10 mg/ml)*
GAPDH Primer - Forward (20 µM)*
GAPDH Primer - Reverse (20 µM)*
8-Well Assay Strips (With 1 Frame)
8-Well Strip Caps
Adhesive Covering Film
F-Spin Column
F-Collection Tube
User Guide

* Spin the solution down to the bottom prior to use.

User Guide & MSDS

[User Guide]*
*Always use the actual User Guide that shipped with your product. Is the above file locked? You can also request user guides by emailing info@epigentek.com along with your contact information and institution name.

Product Citations

Nag M et. al. (May 2018). Histone Modulation Blocks Treg-Induced Foxp3 Binding to the IL-2 Promoter of Virus-Specific CD8⁺ T Cells from Feline Immunodeficiency Virus-Infected Cats. Viruses. 10(6)

Boris Castillo Chabeco et. al. (May 2018). Ethanol-induced modulation of GPR55 expression by human monocyte derived dendritic cells is associated with H4K12 acetylation Alcohol.

Colón-Caraballo M et. al. (February 2018). Effects of histone methyltransferase inhibition in endometriosis. Biol Reprod.

Cascio S et. al. (December 2017). Abnormally glycosylated MUC1 establishes a positive feedback circuit of inflammatory cytokines, mediated by NF-κB p65 and EzH2, in colitis-associated cancer. Oncotarget. 8(62):105284-105298.

Wang YA et. al. (October 2017). T regulatory cell induced Foxp3 binds the IL2, IFNγ, and TNFα promoters in virus-specific CD8+ T cells from feline immunodeficiency virus infected cats. AIDS Res Hum Retroviruses.

Figliozzi RW et. al. (September 2017). Reversing thyroid hormone mediated repression of a HSV-1 promoter via computationally guided mutagenesis. J Cell Sci.

Peng Y et. al. (April 2017). Pdgfrb is a direct regulatory target of TGFβ signaling in atrioventricular cushion mesenchymal cells. PLoS One. 12(4):e0175791.

Zhu B et. al. (March 2016). Transcriptional regulation of miR-15b by c-Rel and CREB in Japanese encephalitis virus infection. Sci Rep. 6:22581.

Gezer U et. al. (December 2015). Histone Methylation Marks on Circulating Nucleosomes as Novel Blood-Based Biomarker in Colorectal Cancer Int J Mol Sci. 16(12):29654-29662.

Zhu M et. al. (January 2015). Alpha-fetoprotein activates AKT/mTOR signaling to promote CXCR4 expression and migration of hepatoma cells. Oncoscience. 2(1):59-70.

Figliozzi RW et. al. (November 2014). Thyroid hormone-dependent epigenetic suppression of herpes simplex virus-1 gene expression and viral replication in differentiated neuroendocrine cells. J Neurol Sci. 346(1-2):164-73.

Li J et. al. (October 2014). Sex-dependent regulation of hepatic CYP3A by growth hormone: Roles of HNF6, C/EBPα, and RXRα Biochem Pharmacol.

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