Overview

  • Product name
    High-Sensitivity ChIP Kit
    See all ChIP Kit kits
  • Sample type
    Tissue, Adherent cells, Suspension cells
  • Species reactivity
    Reacts with: Mammals, Other species
  • Product overview

    High-Sensitivity ChIP Kit (ab185913) 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 High-Sensitivity ChIP Kit can be used for various downstream applications including PCR (ChIP-PCR), microarrays (ChIP-on-chip), and sequencing (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 1-2 x 105 cells or 10-20 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 5-6 x 105 cells or 50 to 60 mg tissues.


     


    Primers


    The GAPDH primers provided with the kit are for the human sequence. If using the kit with a difference species, GAPDH primers for that species will need to be acquired.


     

  • Notes

    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. The High-Sensitivity ChIP Kit is designed to achieve these goals by maximizing sensitivity and minimizing non-specific background signals.

  • Tested applications
    Suitable for: ChIPmore details

Properties

  • Storage instructions
    Please refer to protocols.
  • Components 24 tests 48 tests
    1000X Protease Inhibitor Cocktail 1 x 30µl 1 x 60µl
    8-Well Assay Strips (with Frame) 3 units 6 units
    8-Well Strip Caps 3 units 6 units
    Adhesive Covering Film 1 unit 2 units
    Antibody Buffer 1 x 3ml 1 x 6ml
    Anti-RNA Polymerase II 1 x 8µl 1 x 16µl
    Blocker Solution 1 x 2ml 1 x 4ml
    ChIP Buffer 1 x 6ml 1 x 12ml
    DNA Binding Solution 1 x 7ml 1 x 14ml
    DNA Elution Buffer 1 x 1ml 1 x 2ml
    DNA Release Buffer 1 x 8ml 1 x 16ml
    Enrichment Enhancer 1 x 55µl 1 x 110µl
    F-Collection Tube 30 units 50 units
    F-Spin Column 30 units 50 units
    GAPDH Primer - Forward (20 µM) 1 x 8µl 1 x 16µl
    GAPDH Primer - Reverse (20 µM) 1 x 8µl 1 x 16µl
    Lysis Buffer 1 x 14ml 1 x 28ml
    Non-Immune IgG (1 mg/ml) 1 x 10µl 1 x 20µl
    Proteinase K (10 mg/mL) 1 x 60µl 1 x 120µl
    Rnase A 1 x 30µl 1 x 60µl
    Wash Buffer 1 x 25ml 2 x 25ml
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab185913 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
ChIP Use at an assay dependent concentration.

The GAPDH primers provided with the kit are for the human sequence. If using the kit with a difference species, GAPDH primers for that species will need to be acquired.

Images

  • 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 ab185913 and a quantitative PCR Fast Kit.

  • Sheared chromatin isolated from different numbers of MCF7 cells was used for ChIP-qPCR analysis of ER-a enrichment in TFF1 promoters using ab185913 and a quantitative PCR Fast Kit.

  • Histone H3K18ac ChIP assay was carried out using ab185913.

    ChIP-Seq reads align with the same peak sites as ENCODE data.

Protocols

References

This product has been referenced in:
  • Skucha A  et al. MLL-fusion-driven leukemia requires SETD2 to safeguard genomic integrity. Nat Commun 9:1983 (2018). Read more (PubMed: 29777171) »
  • Dimova LG  et al. Inhibiting Cholesterol Absorption During Lactation Programs Future Intestinal Absorption of Cholesterol in Adult Mice. Gastroenterology 153:382-385.e3 (2017). Read more (PubMed: 28438611) »

See all 3 Publications for this product

Customer reviews and Q&As

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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