Overview

  • Product name
    Methylated DNA Quantification Kit (Fluorometric)
    See all 5-methylcytosine kits
  • 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.

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

Images

  • 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

References

This product has been referenced in:
  • Pinzón-Cortés JA  et al. Effect of diabetes status and hyperglycemia on global DNA methylation and hydroxymethylation. Endocr Connect 6:708-725 (2017). Read more (PubMed: 28993426) »
See 1 Publication for this product

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Clunky kit

Average Below average 2/5 (Ease of Use)
Abreviews
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

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

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

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

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

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

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

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

Sign up