Question (55450) | DNA demethylase (total) Activity Quantification Assay Kit (ab113472)

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Question

Q1: What sample preparation buffer components might interfere with assay?
Q2: Is the detection ALP or HRP-based? Can any endogenous enzyme in the sample "if the nuclear fraction is not purified completely" interfere with the detection? Potentially?
Q3: The capture antibody is 5-methylcytosine antibody according to the Protocol Booklet. Is that correct? Does it have any cross-reactivity towards other modified nucleotides? Has it been tested? Any data?Can I order both capture and detection antibodies separately? Are they poly or monoclonal? What are the epitopes and/or immunogen sequences?
Q4:Are there any preference in the lysis buffer for preparing the sample? (e.g. RIPA, NP-40, etc.) Any specific purification step has to be done for the sample before proceeding to the assay using this kit?
Q4:What does the substrate binding solution contain?
Q5: what is the standard?

Answer

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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