Question (35977) | Histone H3 (K9) Methyltransferase Activity Quantification Assay Kit (ab113453)

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Question

1. How many cells or mg of tissue will yield 4-20ug of nuclear extract?
2. Can customer perform this assay using their own 96-well plate? Maybe after avidin coating?
3. The protocol says to read the absorbance within 2-15 minutes? Would it be detrimental to read the signal after 15 minutes? Does the signal fade or for how much longer is it stable?
4. If a plate reader does not read at 450nm, but at 470nm, will that still work?
5. If a researcher is concerned about low detection: Can an overnight incubation of the nuclear extracts with assay buffer (step 3) be done? Or could another step be done overnight, e.g. the capture antibody incubation?

Answer

Thank you for contacting us.
Here are the answers to your questions:
1. 100,000-500,000 cells or 10-50 mg tissues.
2. It is possible but results are not guaranteed.
3. It can be stable for 30 min after adding stop solution.
4. It may not work at 470 nm.
5. Enzyme or nuclear extract reaction step should be finished within 1-3 h at 37C. Capture antibody step could be overnight at 4C. However the background may be increased.
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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