Overview

  • Product name
  • Sample type
    Adherent cells, Suspension cells
  • Product overview

    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.

  • Notes

    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 
  • Tested applications
    Suitable for: ChIPmore details
    Unsuitable for: CHIPseq

Properties

Applications

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.

Application Abreviews Notes
ChIP Use at an assay dependent concentration.
  • Application notes
    Is unsuitable for CHIPseq.
  • Images

    • 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.

    Protocols

    References

    This product has been referenced in:
    • Islam SS  et al. Eugenol potentiates cisplatin anti-cancer activity through inhibition of ALDH-positive breast cancer stem cells and the NF-?B signaling pathway. Mol Carcinog 57:333-346 (2018). Read more (PubMed: 29073729) »
    • Zhou A  et al. RNA Binding Protein, HuR, Regulates SCN5A Expression Through Stabilizing MEF2C transcription factor mRNA. J Am Heart Assoc 7:N/A (2018). Read more (PubMed: 29678826) »
    See all 41 Publications for this product

    Customer reviews and Q&As

    1-10 of 48 Abreviews or Q&A

    Answer

    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 remain in water solution above the slurry. One benefit of using the slurry extraction is that divalent heavy metals can introduce DNA damage at high temperature (e.g. 100oC) and removing the ions can diminish this concern. In addition, magnesium is necessary for nuclease activation and binding these ions inactivates the enzyme, therefore the DNA will be protected. Elution of DNA from the slurry is achieved through centrifugation in steps 11-13 of part E (DNA purification) in the protocol booklet.

    Read More

    Question

    Hello,

    Thanks for the previous response, I was not able to salvage my protein but I appreciate the help.

    I have another question I was hoping you could help me with. As I have mentioned before, I am using the ChIP kit ab500 to isolate the HIF-1alpha protein from some samples I have. My supervisor has asked me to modify this protocol to allow for a western blot to be preformed with the isolated protein rather than following through with the rtPCR as indicated in the protocol. I was in contact with abcam prior to starting this experiment, they told me they couldn't foresee any issues in altering the protocol for use in a western blot but they would not be able to tell me for sure. I decided to go ahead with my proposed protocol and have had many difficulties which I am trying to overcome.

    One issue I have is that I need to remove the protein from the protein binding beads in order to run the sample through a gel used in the western blot protocol. To do this, I tried incubating the sample (protein, DNA, protein binding bead complex) in an SDS solution at 98C for 10minutes. This method appeared successful at releasing the samples from the beads but I have not obtained results in my experiments. I was wondering if this temperature is too high for the antibody used and if so, what size the free antibody would appear at on a western? I use antibody ab1 for this protocol. My thought is that this incubation temperature and time are too high and therefore the antibody disassociates from the complex and this is why I cannot detect my protein?

    Also, I am questioning whether or not I was able to remove the beads completely from the sample prior to beginning the western blot. If I were to run the beads on the gel, do you know where abouts I would see the corresponding bands and since they too are heated in the incubation, would they degrade and therefore be seen on the gel in the western?

    I have attached my experimental results. As you can see there are not many differences between the samples and negative results. I think the bands differing in the samples and negatives are free antibody that disassociated from the complex during the incubation I discussed above. I am having difficulties determining what all the bands are, and am hoping you might be able to help. Please let me know if you have any thoughts on what all the bands are.

    Read More
    Answer

    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 done this, then I would suggest you try it and make sure the kit is working as it should.

    2 - I have attached the document you sent and I have marked what I think are the heavy and light chains of the antibody (50kDa & 25kDa, respectively). Also, I am not completely familiar with this kit, but is there supposed to be antibody in your negative controls?

    3 - You do not need to heat your samples to 98C to strip the protein from the beads. Heating the sample to 65/70C for 5 minutes is usually enough.

    4 - If you have not removed all of the beads from your sample then they are generally trapped at the top of the gel and do not migrate.

    Please let me know if there is anything else I can help you with.

    Read More

    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.

    Abcam user community

    Verified customer

    Submitted Jun 04 2018

    Answer


    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 vary depending on the type of bacteria. An enzymatic treatment (for example a lyzozyme treatment) may be added to the protocol in order to help the cell lysis. You can find two publications in attachment for some guidelines to perform a lyzozyme treatment for bacteria lysis before ChIP.

    It is also recommended to check the efficiency of the shearing on agarose gel before starting the immunoprecipitation to ensure that the starting material for the IP is of good quality.

    So the best would be to test the addition of a lyzozyme treatment to the protocol and to perform a time course for the shearing to determine the optimal conditions. For the shearing time, it is always recommended to select the shorter timing resulting in a good shearing profile.

    Since there is an antibody against Histone H3 is provided as a positive control with that kit and bacteria do not express this protein (Peter J. Rizzo (2003). http://www.nature.com/cr/journal/v13/n4/full/7290166a.html) some bacteria specific positive control might need to be established.

    Publications:
    Al-Bassam et al., 2014 PMID: 25101778 [PubMed]
    Wang H. et al., 2012 PMID: 22194453 [PubMed]

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    Question
    Answer

    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.

    Read More

    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.

    Mr. Christian Marx

    Verified customer

    Submitted Jan 08 2014

    Answer

    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.

    Read More

    Answer

    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.

    Read More

    Answer

    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 adding it to the samples to make sure the correct amount of SDS is transfered.

    The cooling seems to be more effective when using the water bath than using ice and therefore the SDS fell out in the samples. This is not a problem is the samples are brought back to RT after sonification and before the next step in the protocol.

    I hope this information is helpful and wish you good luck with your research.

    Read More

    Answer

    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.

    If you need any further assistance in the future, please do not hesitate to contact me.

    Read More

    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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