Overview

  • Product name

    Universal Methyltransferase Activity Assay Kit
    See all Methyltransferase kits
  • Detection method

    Fluorescent
  • Sample type

    Purified protein
  • Assay type

    Enzyme activity
  • Product overview

    Universal Methyltransferase Activity Assay Kit (ab139433) is a homogeneous mix-and-read fluorescent assay for the determination of any S-adenosyl-L-methionine (AdoMet/SAM) dependent methyltransferase activity. It is suitable for end-point or kinetic read options, which is ideal for determining mechanism of action, kinetics, and screening candidate compounds. The assay is amendable to HTS and miniaturization.

    ab139433 is a complete kit for the screening of candidate compounds that may alter normal methyltransferase activity.

  • Notes

    Methylation of proteins, nucleic acids and oligosaccharides is an important post- translational regulatory event. Activities that are methylation-related include meiosis, biosynthesis, development, signal transduction, chromatin regulation, and gene silencing. The enzymes that mediate the covalent transfer of a methyl group from a donor to an acceptor molecule are methyltransferases. Methyltransferases have structurally unrelated acceptors as diverse as proteins and DNA, however frequently use S -adenosylmethionine as a universal donor. Part of the acceptor diversity of this enzyme family relates to the flexible structural folds that bind these molecules in proximity of the donor. The side- chains of lysine, arginine, glutamate, glutamine, asparagines, and isoprenylated residues serve as methylation sites in proteins like histones. Changes in methylation patterns have been tightly linked to disease states such as cancer and vascular disease.

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests
    Black Microtiter Plate 1 unit
    Methyltransferase Positive Control 1 x 0.2ml
    Methyltransferase Reaction Buffer Concentrate 1 x 0.3ml
    Plate Sealer 1 x 3 units
    Transferase Assay Buffer Concentrate 1 x 15ml
    Transferase Detection Solution Concentrate 1 x 0.15ml
  • Research areas

Images

  • Using SET7/9 and TAF-10 as an enzyme substrate system, percent inhibition for dilutions of 5’deoxy-5’(methylthio)-adenosine was tested.
  • 0.5µM SET7/9 was tested with 15µM TAF-10 substrate in the kinetic assay format. Mean relative fluorescence was plotted against the stop time interval.
  • Titration assay of an engineered portion of human lysine specific histone methyltransferase SET7/9 using 15µM of the peptide substrate TAF-10. Serial dilutions of the enzyme were prepared in Transferase Assay Buffer. Average relative fluorescence was plotted against SET7/9 concentration to generate the following graph. This is for maximum signal within the detection range of the plate reader, with a signal to noise ratio sufficient for easy detection of altered enzyme activity.

Protocols

References

ab139433 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

Answer

As we discussed over the phone, I have looked into whether the Universal Methyltransferase Activity Assay Kit (ab139433) is likely to be suitable for the experiments you have been planning. The kit was developed to allow for the methyltransferase activity to be measured for biochemical assays using purified methyltransferase. The end user has to find their own enzyme/substrate to use in the assay.

It is theoretically possible to use the kit with biological samples however there are likely to be a number of problems encountered in this. You would not be able to target one enzyme as most substrates can be used with a variety of different methyltransferases (i.e. it would react with multiple enzymes in the sample). You would also need to optimize the cell culture samples as if there were the enzyme source and determine the optimal enzymatic kinetics required to get optimal window of activity when used with the substrate of your choice. However, any reductants will be an issue including DTT or any thiol containing sample/reagent as they will interfere with the assay.

Therefore we would not advise using this assay for biological samples. Unfortunately we do not currently have any alternative assay which would work with biological samples, appart form if you are particularly interested in the methyltransferase activity on Histon H3 K4, K9 or K27 (kits ab113452, ab113453 and ab113454).

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