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We have now performed a repeat of the demethylase activity assay using the kits you supplied. However, there seems to be an issue with the buffer you recommend for use with the kit. All of the samples including the buffer only (no cells) wells give a strong positive result, so it appears that the buffer you recommend is interfering with the assay.
Ana is away at the moment but I have cc’d her in this email so she can give you more details about the assay conditions, and perhaps you might want to perform some assays yourself to check the kits.
Asked on Mar 12 2012
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/index.html?datasheet=113467 (or use the following: https://www.abcam.com/index.html?datasheet=113467).
ab113468 EpiSeeker DNMT activity Quantification Kit (Fluorometric)
https://www.abcam.com/index.html?datasheet=113468 (or use the following: https://www.abcam.com/index.html?datasheet=113468).
These two kits measure general methyltransferase activity
We also have available specific assays for:
DNMT1 - ab113469
https://www.abcam.com/index.html?datasheet=113469 (or use the following: https://www.abcam.com/index.html?datasheet=113469).
DNMT3A - ab113470
https://www.abcam.com/index.html?datasheet=113470 (or use the following: https://www.abcam.com/index.html?datasheet=113470).
DNMT3B - ab113471
https://www.abcam.com/index.html?datasheet=113471 (or use the following: https://www.abcam.com/index.html?datasheet=113471).
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.
- 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)
Answered on Mar 12 2012