DNA demethylase (total) Activity Quantification Assay Kit (ab113472)

Thank you for contacting us. The substrate for ab113472 is double stranded DNA. We currently only have the information related to use of this kit with DNA from mammalian tissue/cells. However, based on the working principle, this kit should also work on plant samples.

I hope this helps, please let me know if you need any additional information or assistance.

Thank you for contacting us.
The kit ab113472 is designed to detect DNA demethylase activity and will detect the this activity using a unique methylated DNA substrate. The detection is then colorimetric and can be measured by optical density measure at 450nm. I do not think that this is adapted for measuring LSD1 activity.
We do however have different other kits, which might correspond more to what you are looking for:
We do have the EpiSeeker KDM1/LSD1 activity quantification kit which comes either colorimetric (ab113459) or fluorometric (ab113460).  This kit does measure the activity of the LSD1 by using the di-methylated histone H3-K4 as a substrate for LSD1. The fluorimetric assay is read at Ex/Em = 530/590 nm.
https://www.abcam.com/KDM1LSD1-Activity-Quantification-Assay-Kit-Colorimetric-ab113459.html
https://www.abcam.com/KDM1LSD1-Activity-Quantification-Assay-Kit-Fluorometric-ab113460.html
Alternatively we do also have the EpiSeeker Histone Demethylase KDM1/LSD1 Activity Quantification Assay Kit ab113457. This kit does detect the histone demethylase by indirect detection. https://www.abcam.com/Histone-Demethylase-KDM1LSD1-Activity-Quantification-Assay-Kit-ab113457.html
It might be interesting to note that we do have a website dedicated to the EpiSeeker products. Please feel free to browse this site as well. www.abcam.com/episeeker
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Thank you for contacting us.

Glycerol, Triton X-100 etc might interfere with the kit components. ab113474 is the optimized kit for sample preparation.

The detection is HRP-based and will not interfere by other enzymes/proteins.

The capture antibody is indeed anti 5-methylcytosine antibody and within the indicated concentration range of the sample DNA, it will not cross-react with other modified nucleotides including 5-hmC and 5-OhdG, 5-fC and 5-caC. The capture and detection antibodies are mouse monoclonals and they can be bought separately. The epitope for these antibodies is proprietary information.

RIPA or NP-40 buffer lysates may not be suitable for this kit as these lysates may not keep the enzyme active.

The Substrate binding solution contains Tris and salts.

The standard is synthesized methylated oligos

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Thank you for contacting us.

The compatible kit for sample preparation is ab113474. The chemicals in this kit are selected in such a way that these do not interfere with ab113472.

https://www.abcam.com/EpiSeeker-Nuclear-Extraction-Kit-ab113474.html

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Thank you once again for your enquiries. I very much appreciate your patience waiting for a response.

I have been in touch with the originator of the buffer and the kit. In conclusion, they have retested and have established that using the buffer suggested to prepare the samples meant the kit did not work so well. They have now modified the kit and protocol accordinglyand have retested them togethersuccessfully.

As this means they have included an extra component to the kit, and modified the kit protocol slightly so it can be used with samples prepared with the buffer. We are currently in the process of reviewing any extra MSDS that may be required, as well as arranging restocking anduploading of the new protocol to the online datasheet. The kit has been unpublished temporarily while this is done.

We would of course like to provide some compensation to you, and apologize for the inconvenience this has caused. Therefore, the options we can provide for you are as follows:

1. To provide a full refund on the kits and buffer previously purchased.
Alternatively:
2. To provide a credit note for the kits and buffer previously purchased
Alternatively:
3. We can provide you with free of charge replacements of the kits and buffer once the changes have been completed. This may take a couple of weeks.

I hope this provides a satisfactory outcome for you. I look forward to hearing from you with details of how you would like to proceed.

Thank you for taking the time to contact us.Your email has been forwarded to me as I was originally dealing with this case. I am sorry to hear your colleague Ana is stillhaving difficulty obtaining satisfactory results from this kit

I would like to reassure you that this kit is tested and covered by our 6 month guarantee for. In the event that a product is not functioning in the tested applications and species cited on the product data sheet (and the problem has been reported within 6 months of purchase), we will be pleased to provide a credit note, free of charge replacement or refund.

I would like to investigate this particular case further for you, and also obtain more information for our quality monitoring records. In order to proceed with this, I would appreciate if Ana could provide the information requested in the questionnaire send with my last email. It will help you put the information we require together very easily. I have copied this below. I would appreciate if you could also providesome datawhich would help us to assess the results.

With regards to the buffer, please confirm which bufferyou are referring to? Reviewing the previous correspondence, we recommended to use EpiSeeker Nuclear Extraction Kit (ab113474) for preparing nuclear extracts from cultured cells. Is this the buffer you have been using unsuccessfully? Please confirm how you think it may be interfering with the assay.

With regards to retesting, this is regrettably not something that can always be arranged. However,Iwould like to reassure you that we monitor feedback closely on a weekly basis and we are not currently concerned about the general quality of thiskit or this batch.

In addition to this, we have reviewed the catalog again and found some methylase kits to suggest as requested in Ana's last email. I am sorry these were missed on the previous search:

Methyl transferaseactivity kits:

ab113467 - EpiSeeker DNMT activity Quantification kit (Colorimetric)
https://www.abcam.com/DNMT-Activity-Assay-Kit-Colorimetric-ab113467.html (or use the following: https://www.abcam.com/DNMT-Activity-Assay-Kit-Colorimetric-ab113467.html).

ab113468 EpiSeeker DNMT activity Quantification Kit (Fluorometric)
https://www.abcam.com/DNMT-Activity-Assay-Kit-Fluorometric-ab113468.html (or use the following: https://www.abcam.com/DNMT-Activity-Assay-Kit-Fluorometric-ab113468.html).

These two kits measure general methyltransferase activity

We also have available specific assays for:

DNMT1 - ab113469
https://www.abcam.com/DNMT1-Assay-Kit-ab113469.html (or use the following: https://www.abcam.com/DNMT1-Assay-Kit-ab113469.html).

DNMT3A - ab113470
https://www.abcam.com/DNMT3A-Assay-Kit-ab113470.html (or use the following: https://www.abcam.com/DNMT3A-Assay-Kit-ab113470.html).

DNMT3B - ab113471
https://www.abcam.com/DNMT3B-Assay-Kit-ab113471.html (or use the following: https://www.abcam.com/DNMT3B-Assay-Kit-ab113471.html).

I hope this will be helpful. Thank you for your time and cooperation. We look forward to receiving the completed questionnaire and data.


A. General Information
- Catalogue number:

- Lot number:

- Purchase order number/ order date or preferably the Abcam order number:

- How was the kit stored?


B. Description of the problem
- Are the signals you obtained with control or standard satisfactory? (Yes/No);


- Are the sample readings as expected or inconsistent?


- Are the blank readings as expected?

- Other (e.g. missing components)


C. What do you think the problem is caused by? E.g. enzyme is faulty, developer is faulty or other component that you think might be faulty.



D. Sample
- Sample Type (e.g. Cell culture supernatant, Cell lysates, Tissue lysates or Serum samples)


- Sample Species (e.g. human, mouse or rat)


- Storage (at what temperature and for how long were the samples stored?)

- Sample treatment, if any



- Sometime proteins in samples interfere with readings. Have you removed proteins?


- Sample preparation


E. Protocol used:
Did you follow the protocol exactly as given on the datasheet?


F. Optimization attempts (problem solving); please describe any deviations from the protocol.



G. Have you used the same kit successfully before?


H. Do you obtain the same results every time?


I. Additional Notes; (any additional information you would like to provide)



J. Results:

Thank you for your recent telephone enquiry. I am sorry to hear you have some concerns regarding the results from this kit. In answer to yourquestions: 1. We are obtaining higher readings from our control that our samples, please explain how we can calculate the DNA methylase activity in this case. (control is bright yellow, samples less yellow). It is expected that OD readings in the enzyme-free control wells are higher than that in the sample wells. A simple calculation of total DNA demethylase activity can be carried out withuse the first formula in the protocol provided with the kit: https://www.abcam.com/ps/products/113/ab113472/documents/ab113472%20EpiSeeker%20DNA%20demethylase%20(Total)%20Activity%20Quantification%20Assay%20Kit%20(Website).pdf 2. Working principle: In an assay with this kit, the unique methylated DNA substrate is stably captured on the strip wells. Active DNA demethylases bind to and demethylate DNA substrate. The methylated DNA can be recognized with a high affinity 5-methylcytosine antibody. The ratio or amount of methylated DNA, which is inversely proportional to enzyme activity, can then be colorimetrically quantified through an ELISA-like reaction. I hope this will help to explain the results you have. However, as discussed on the telephone, if you still have some concerns, please do not hesitate to contact me again. I woudl appreciate in this case if you could send an attached spreadsheet of the results (with a full description of each sample) and also complete the questionnaire below. This will provide us with the information we require to investigate this case. I would like to reassure you that the kit is covered by our 6 month guarantee. We can provide a free of charge replacement, credit note or refund if it is not working as described on the datasheet. 3. What do the buffers contain? The assay buffercontains Tris-HCl and Mg salt. 4. EpiSeeker Nuclear Extraction Kit (ab113474) can be used for preparing nuclear extracts from cultured cells. https://www.abcam.com/Nuclear-Extraction-Kit-ab113474.html (or use the following: https://www.abcam.com/Nuclear-Extraction-Kit-ab113474.html). 5. Can we have a discount for methylase activity kit? I am sorry to confirm we do not have methylase activity kits in the catalog at present. I hope this will be helpful to you. If you have any further questions orconcerns, please do not hesitate to contact me. QUESTIONAIRE: A. General Information - Catalogue number: - Lot number: - Purchase order number/ order date or preferably the Abcam order number: - How was the kit stored? B. Description of the problem - Are the signals you obtained with control or standard satisfactory? (Yes/No); - Are the sample readings as expected or inconsistent? - Are the blank readings as expected? - Other (e.g. missing components) C. What do you think the problem is caused by? E.g. enzyme is faulty, developer is faulty or other component that you think might be faulty. D. Sample - Sample Type (e.g. Cell culture supernatant, Cell lysates, Tissue lysates or Serum samples) - Sample Species (e.g. human, mouse or rat) - Storage (at what temperature and for how long were the samples stored?) - Sample treatment, if any - Sometime proteins in samples interfere with readings. Have you removed proteins? - Sample preparation E. Protocol used: Did you follow the protocol exactly as given on the datasheet? F. Optimization attempts (problem solving); please describe any deviations from the protocol. G. Have you used the same kit successfully before? H. Do you obtain the same results every time? I. Additional Notes; (any additional information you would like to provide) J. Results: