製品の概要

  • 製品名
  • サンプルの種類
    Adherent cells, Suspension cells
  • 製品の概要

    ChIP kit ab500 provides a protocol and reagents for running ChIP assays including:
    - cell lysis and chromatin extraction
    - chromatin shearing and DNA fragment length analysis
    - immunoprecipitation and DNA purification


    DNA produced using the kit can be analyzed using qPCR.


    The kit has been validated for ChIP assays with mammalian samples.

  • 特記事項

    This kit uses Protein A sepharose beads for antibody pulldown. See table below for Protein A and Protein G binding affinities with antibodies from commonly used species. For other species of antibody, consult the table in the protocol booklet.

    Species raised in

    Isotype  Protein A binding affinity Protein G binding affinity
           
    Rabbit  All isotypes +++ ++
           
    Goat All isotypes - ++
           

     

     

    Mouse   

    IgG1  + +++ 
    IgG2a  +++ +++
    IgG2b  ++ ++ 
    IgG3  +  +
    IgM  Use anti-mouse IgM 
  • アプリケーション
    適用あり: ChIPmore details
    適用なし: CHIPseq

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab500 in the following tested applications.

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

アプリケーション Abreviews 特記事項
ChIP Use at an assay dependent concentration.
  • 追加情報
    Is unsuitable for CHIPseq.
  • 画像

    • Chromatin immunoprecipitation assay was performed to define the interaction of serum response factor (SRF) with the intragenic serum response element (SRE) regulatory motif in mouse using ab500 ChIP Kit

    • Chromatin immunoprecipitation using ab500 ChIP Kit and Histone H3 (tri methyl K9) antibody (ab8898). Chromatin was prepared from HeLa cells using the Abcam ChIP kit protocol. Cells were fixed with formaldehyde for 10 min. ChIP was performed with 2 µg of ab8898 (blue). No antibody was added to the beads control (yellow). Immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active/inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of transcribed region.
    • Chromatin immunoprecipitation using ab500 ChIP Kit and Histone H3 (tri methyl K4) antibody (ab12209). Chromatin was prepared from HeLa cells using the Abcam ChIP kit protocol. Cells were fixed with formaldehyde for 10 min. ChIP was performed with 5 µg of ab12209 (blue). No antibody was added to the beads control (yellow). Immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active/inactive loci). Primers and probes are located in the first kb of the transcribed region.
    • Chromatin immunoprecipitation using ab500 ChIP Kit and Histone H3 antibody (ab1791). Chromatin was prepared from HeLa cells using the Abcam ChIP kit protocol. Cells were fixed with formaldehyde for 10 min. ChIP was performed with 2 µg of ab1791 (blue). No antibody was added to the beads control (yellow). Immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active/inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

    プロトコール

    参考文献

    This product has been referenced in:
    • Lorentzen A & Mitchelmore C NDRG2 gene copy number is not altered in colorectal carcinoma. World J Clin Oncol 8:67-74 (2017). ChIP ; Human . Read more (PubMed: 28246586) »
    • Albert BJ  et al. Combinations of isoform-targeted histone deacetylase inhibitors and bryostatin analogues display remarkable potency to activate latent HIV without global T-cell activation. Sci Rep 7:7456 (2017). Read more (PubMed: 28785069) »

    See all 36 Publications for this product

    レビューと Q&A

    DNA purification slurry is often used for DNA extraction. The extraction is set up under aqueous alkaline conditions. In this environment, the slurry has an increased affinity for heavy metal cations such as Ca2+, Mn2+, and Mg2+. Nuclear DNA and RNA re...

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    Thank you for your reply.

    I will do my best to see if I can help you:

    1 - When you analyze your results via PCR rather than western blotting, do you get the correct results (eg no signal in the negative control)? If you have not don...

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    ab500 ChIP, Runx2 binding to target promoters

    Excellent Good 4/5 (Ease of Use)
    Abreviews
    Chromatin immunoprecipitation. ab500 ChIP Kit (abcam) was used according to the manufacturer’s instructions. Briefly, VSMCs were trypsinsed, centrifuged and then washed in ice-cold PBS. Cells were centrifuged again and then fixed in 1.1% Formaldehyde in PBS. Reactions were quenched with glycine and cells were washed in ice cold PBS before being lysed. Chromatin was sheared to approximately 500-1000 bp fragments using a sonicator at 4 °C. Chromatin was diluted and input chromatin was collected. Remaining chromatin was used for ChIP using 4 ug Runx2 (D130-3) as antibody of interest, 4 ug anti-histone H3 as a positive control and no antibody as a negative control. Antibodies were added for 12 hours at 4 °C and then Protein A sepharose beads were used to precipitate protein/DNA complexes. Cross-links were reversed by heating at 98 °C followed by Proteinase K addition and DNA purification. Samples were analysed by pPCR.
    Username

    Abcam user community

    Verified customer

    投稿 Jun 04 2018


    The use of ab500 ChIP kit has not been validated so far on bacteria.

    The part of the protocol that may need some optimization is the chromatin preparation. Actually, lysis of bacteria is more complex than lysis of eukaryotic cells and may...

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    the concentration of formaldehyde needed for the kit is actually 1% but you could use any percentage below 4%. 1% is just what was used in our lab.

    ChIP Assay in HeLa cells

    Good Average 3/5 (Ease of Use)
    Abreviews
    Experimental set up:
    HeLa cells treated with 1 µM Dexamethasone (Dex) for 6h.
    IP was performed with 4.0 µg rabbit anti-GR antibody (H-300, Santa Cruz).

    Recommendations for product and protocol optimization:
    - Step A2
    Spin cells for 5 min instead of 10 min at 500 g at 4°C (not RT) for saving time and to avoid aberrant DNA interactions that might occur if time between harvest and cross linking is too long.

    - Step B8
    Using a sonication device with a sounding rod with a total lysate volume of 100 uL (3 ChIP assays) is difficult to handle. As a consequence, you easily lose much sample by dispersion. Better incubate cell pellet in the indicated 100 uL buffer D/PI mix for 5 min on ice and add 1X ChIP buffer (containing PI) to a volume of 350 uL. After sonication, add 580 uL 1X ChIP buffer (incl. PI) to the lysate (step D2). From this step the total volume for each sample will be as indicated in the manual. Sonication of bigger sample volume will not compromise the outcome of your experiment!

    - Step B11
    Spin sonicated lysates for 10 min instead of 5 min at 14,000 g at 4°C.

    General remark:
    Switching temperatures from spinning to RT at step A2 and A3 does not change the experimental results. Better keep 4°C constantly.
    Username

    Mr. Christian Marx

    Verified customer

    投稿 Jan 08 2014

    It is possible to freeze the sheared chromatin and resume the ChIP at a later time, but once the ChIP has started, it is not possible to stop.

    The kit is for 24 assays. It is 100 µl per ChIP. With 3000 µl DNA purifying slurry, you can do 30 ChIPs. But you are correct, you have to also use 100 µl per INPUT. So with this kit, you can do 24 assays + 6 INPUT.

    Thank you for your inquiry.

    I am happy to confirm that it is normal to have precipitation at 4°C in buffer D. This is due to the SDS as you said in your email.

    It is important to bring the buffer to room temperature before addin...

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    Thank you for your enquiry regarding ab500.

    We never tested this kit on FUNGI chromatin. You may need to optimize first the shearing conditions. Afterwards, if you use a specific antibody to FUNGI chromatin, the kit will probably work.
    <...

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    1-10 of 48 Abreviews or Q&A

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

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