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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). How does this kit work? What do the buffer contain? What extraction reagents would be suitable to prepare cell culture samples (eg MCA, MB231 cell lines). Need a concentrated solution as only 2 ul used per test. Can we have a discount for methylase activity kit?
Asked on Jan 10 2012
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/index.html?datasheet=113474 (or use the following: https://www.abcam.com/index.html?datasheet=113474). 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:
Answered on Jan 10 2012