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Epigenetics and Nuclear Signaling DNA methylation Methylated DNA
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Methylated DNA Quantification Kit (Fluorometric) (ab117129)

  • Datasheet
  • SDS
  • Protocol Booklet
Reviews (1)Q&A (6)References (1)

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Functional Studies - Methylated DNA Quantification Kit (Fluorometric) (ab117129)

    Key features and details

    • Assay type: Quantitative
    • Detection method: Fluorescent
    • Platform: Microplate reader
    • Assay time: 4 hr
    • Sample type: Adherent cells, Plasma, Saliva, Suspension cells, Tissue, Urine
    • Sensitivity: 50 pg/well

    You may also be interested in

    Kit
    Product image
    Hydroxymethylated DNA Quantification Kit (Fluorometric) (ab117131)
    ChIP
    Product image
    Methylated DNA Immunoprecipitation (MeDIP) Kit - DNA (ab117133)

    View more associated products

    Overview

    • Product name

      Methylated DNA Quantification Kit (Fluorometric)
      See all 5-methylcytosine kits
    • Detection method

      Fluorescent
    • Sample type

      Saliva, Urine, Plasma, Tissue, Adherent cells, Suspension cells
    • Assay type

      Quantitative
    • Sensitivity

      >= 50 pg/well
    • Assay time

      4h 00m
    • Species reactivity

      Reacts with: Species independent
    • Product overview

      DNA methylation is a covalent modification of the cytosine ring at the 5' position, resulting in 5-methylcytosine (5-mC). In somatic cells, 5-mC is found almost exclusively in the context of paired symmetrical methylation of the dinucleotide CpG. The biological important of 5-mC as a major epigenetic modification in phenotype and gene expression has been recognized widely. Quantification of 5-mC content or global methylation status in cells such as tumor cells could provide very useful information for detection and analysis of cancer.

      Abcam's Methylated DNA Quantification Kit (Fluorometric) (ab117129) is a complete set of optimized buffers and reagents to quantify global DNA methylation by specifically measuring levels of 5-methylcytosine (5-mC) in a microplate-based format.

      It can be paired to use with Hydroxymethylated DNA Quantification Kit (Fluorometric) (ab117131), which specifically quantifies 5-hmC, for simultaneously quantification of both methylated and hydroxymethylated DNA.

    • Platform

      Microplate reader

    Properties

    • Storage instructions

      Please refer to protocols.
    • Components 48 tests 96 tests
      10X Wash Buffer 1 x 14ml 1 x 28ml
      8-Well Assay Strips (with Frame) 6 units 12 units
      Binding Solution 1 x 5ml 1 x 10ml
      Capture Antibody, 1000 µg/mL 1 x 4µl 1 x 8µl
      Detection Antibody, 400 µg/mL 1 x 8µl 1 x 16µl
      Enhancer Solution 1 x 8µl 1 x 16µl
      Fluoro Developer 1 x 8µl 1 x 16µl
      Fluoro Dilutor 1 x 4ml 1 x 8ml
      Fluoro Enhancer 1 x 8µl 1 x 16µl
      Negative Control, 20 µg/mL 1 x 10µl 1 x 20µl
      Positive Control, 20 µg/mL 1 x 10µl 1 x 20µl
    • Research areas

      • Epigenetics and Nuclear Signaling
      • DNA methylation
      • Methylated DNA
      • Kits/ Lysates/ Other
      • Kits
      • Epigenetic kits
      • DNA modification and methylation
    • Alternative names

      • 5 mC
      • 5 MethylCytosine
      • 5mC

    Associated products

    • Related Products

      • Methylated DNA Quantification Kit (Colorimetric) (ab117128)
      • Hydroxymethylated DNA Quantification Kit (Colorimetric) (ab117130)
      • Hydroxymethylated DNA Quantification Kit (Fluorometric) (ab117131)

    Images

    • Functional Studies - Methylated DNA Quantification Kit (Fluorometric) (ab117129)
      Functional Studies - Methylated DNA Quantification Kit (Fluorometric) (ab117129)
      Demonstration of high sensitivity and specificity of methylated DNA detection achieved using ab117129. Synthetic unmethylated DNA (contains 50% of cytosine) and methylated DNA (contains 50% of 5-methylcytosine) were added into the assay wells at different concentrations and then measured with ab117129.

    Protocols

    • Protocol Booklet

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet
    • SDS
  • References (1)

    Publishing research using ab117129? Please let us know so that we can cite the reference in this datasheet.

    ab117129 has been referenced in 1 publication.

    • Pinzón-Cortés JA  et al. Effect of diabetes status and hyperglycemia on global DNA methylation and hydroxymethylation. Endocr Connect 6:708-725 (2017). PubMed: 28993426

    Customer reviews and Q&As

    Show All Reviews Q&A
    Submit a review Submit a question

    1-7 of 7 Abreviews or Q&A

    Clunky kit

    Average Below average 2/5 (Ease of Use)
    Abreviews
    Abreviews
    abreview image
    I usually use molecular techniques for global methylation (LUMA, etc.) but wanted to give this kit a try since I was hoping it would be easier. Yes, the assay can be done in a day but it's somewhat clunky and has many many steps. You're out of luck if you don't have a multichannel pipette since there are alot of incubation/wash steps... Also, I wasn't too happy with my standard curve.
    In brief, perhaps this is good if you're new to methylation assays, but if you know how to measure methylation without a kit, it might actually be easier.

    Abcam user community

    Verified customer

    Submitted Aug 16 2018

    Question

    The controls with known concentrations had inconclusive signals and even my sample had a bigger signal in the well with the lowest quantity of DNA. We read the plate using two different plate readers and in none of them it worked properly. After that we also removed the Fluoro-Development Solution from the wells and we added more 10microL of this solution to try to get a correct signal but this also did not work (in this measurement the signal of the negative control was bigger than all the others).

    What do you think that could be the problem and how can we have correct results?

    Read More

    Abcam community

    Verified customer

    Asked on Jul 25 2014

    Answer


    Having reviewed this case with the lab, I would like to offer some suggestions to help optimize the results from ab117129.

    It appears that the signal may be saturated either due to

    1) insufficient washing or
    2) too much time between adding Fluoro-Development Solution and reading the plate.

    We have the following recommendations to improve the assay results:

    1) add an additional wash at each wash step making sure to remove all residual wash solution from the wells at each step and especially before the signal detection step using a multi-channel pipette; and
    2) reduce the color development time after adding the Fluoro-Development Solution.

    The color development is continuous and cannot be stopped therefore after some time the signal will reach plateau for all of the wells. In general, the best time for measuring fluorescence should be 2-3 min after adding Fluoro-Development Solution, or based on the color change. When a slight pink color is seen in the wells containing the highest amount of the positive control and the blank well is still clear, the measurement should be carried out.

    Read More

    Elisa Thomas

    Abcam Scientific Support

    Answered on Jul 25 2014

    Question

    Hello, I received this query via Facebook, and would like to know how best to answer it? Any help is much appreciated.

    · I got the episeeker methylated DNA Quantification and would like to know the constitution of TE buffer specified in protocol.
    Thanks for the time available,


    Best Regards,

    Read More

    Abcam community

    Verified customer

    Asked on Dec 14 2012

    Answer

    Thank you for your enquiry and interest regarding ab117129.
    I can confirm that the TE buffer specified in the protocol is constituted with 10 mM Tris-HCl pH 7.5-8 and 1 mM EDTA.
    If you need any further assistance in the future, please do not hesitate to contact me.

    Read More

    Abcam Scientific Support

    Answered on Dec 14 2012

    Question

    Does the assaydetect single strand DNA or double strand DNA? If both, is it more sensitive for detection of methylated single or double stranded?

    Read More

    Abcam community

    Verified customer

    Asked on Jul 23 2012

    Answer

    Thank you for contacting Abcam.


    Yes, the assay detect 5-mC on both single and double stranded DNA with the nearly same sensitivity. The standard is double stranded DNA.



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

    Read More

    Abcam Scientific Support

    Answered on Jul 23 2012

    Question

    Thank you very much for your help. It is good to know that the excitation wavelength was fine. However, we decided to stop working with these assays – at least for the moment.
    I appreciate the time you have spent but would it be possible to go for a refund, like your colleague wrote?
    Best regards,

    Read More

    Abcam community

    Verified customer

    Asked on Jun 15 2012

    Answer

    Thank you for contacting us.

    Your credit note ID is ******.

    I am sorry that this kit did not perform as stated on the datasheet, I have asked our Finance department to issue a credit note for you. The credit note may be used in one of the following ways:

    (1) Redeemed against the original invoice if this hasn't already been paid.
    (2) Held on the account for use against a future order.
    (3) A full refund can be offered where no other invoices are outstanding.

    Please contact your Finance department to confirm how you would like the credit note to be used and ensure it is not redeemed without your knowledge.

    To specifically receive a refund please ask your Finance department to contact our Finance department at creditcontrol@abcam.com or by telephone using the information at the “Contact Us” link in the top right corner of our website.

    The credit note ID is for your reference only, please refer to the credit note ID in any correspondence with our accounting department. We will send you the completed credit note by email or postal mail with the actual credit note number which will start with the letters CGB.

    I hope this experience will not prevent you from purchasing other products from us in the future. Our Scientific Support team is always at your service should you require further expert advice

    Read More

    Abcam Scientific Support

    Answered on Jun 15 2012

    Question

    To whom it may concern,
    I have some problems/questions regarding the following products:
    ab117129 EpiSeeker Methylated DNA Quantification Kit (Fluorometric), Lot GR82221-2
    ab117131 EpiSeeker Hydroxymethylated DNA Quantification Kit (Fluorometric), Lot GR83043-1
    Both assays I performed with the standard curve prepared according to the respective manual but I saw inconsistencies in these curves. Some values in that dilution series absolutely do not fit (while others are ok) and in some cases even the replicate values differ strongly.
    The replicates of my samples differ as well - in some cases up to 30%.
    I measured with Ex/Em 550/595 nm. Apart from that I strictly followed the manual.
    Do you have any ideas or hints?
    Thank you very much in advance!

    Read More

    Abcam community

    Verified customer

    Asked on Jun 14 2012

    Answer

    Thank you very much for your patience.

    We have now received the stimulation graph from the laboratory. Please find it in the attachment. Based on that graph, I am happy to confirm that stimulation at 550nm is absolutely fine, as well as emission reading at 595nm.I hope this information is helpful and does reassure you.

    In regards to the inconsistency in your results, I would be pleased to investigate this further. Can you therefore please send me the raw annotated data as well as detailled information on the samples andtheir preparation? Often inconsistency in duplicates are due to technical problems, such as unsufficient washing, bubbles in the well orsmall pipetting difference.I will however be pleasedto look into this problem further with the helpof the data from you.

    I look forward to hear back from you. Thank you for your cooperation.

    Read More

    Abcam Scientific Support

    Answered on Jun 14 2012

    Question

    To whom it may concern,
    I have some problems/questions regarding the following products:
    ab117129 EpiSeeker Methylated DNA Quantification Kit (Fluorometric), Lot GR82221-2
    ab117131 EpiSeeker Hydroxymethylated DNA Quantification Kit (Fluorometric), Lot GR83043-1
    Both assays I performed with the standard curve prepared according to the respective manual but I saw inconsistencies in these curves. Some values in that dilution series absolutely do not fit (while others are ok) and in some cases even the replicate values differ strongly.
    The replicates of my samples differ as well - in some cases up to 30%.
    I measured with Ex/Em 550/595 nm. Apart from that I strictly followed the manual.
    Do you have any ideas or hints?
    Thank you very much in advance!

    Read More

    Abcam community

    Verified customer

    Asked on Jun 11 2012

    Answer


    I would like to inform you that we are still investigating the problem you experienced with the kits. The lab has already provided the following information: "The reading can be carried out at 530/590 nm +/- 10 generally. Thus, emission at 595 nm is fine, and excitation at 550 may be Ok but suboptimal". From this Claudiarequested a graph of the excitiation and emission of the fluorochrome used in the kits and we are still waiting for more details. I have sent a reminder to the lab, we are very sorry we cannot still answer this question and I hope we will be able to do so within the next couple of days.

    I appreciate the time you have spent on these experiments and would be pleased to arrange a replacement, a refund or credit note in compensation.As a replacement, please note thatthe only lots (for both kits)we currently have in stock are the same as the ones your received, let me know if you would like to receivefree of charge replacements and I will ask the source of the kits ifalternative lotsare available.

    I look forward to hearing from you with details of how you would like to proceed.

    Read More

    Abcam Scientific Support

    Answered on Jun 11 2012

    Please note: All products are "FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES"
    For licensing inquiries, please contact partnerships@abcam.com

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