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HTRF Anti histone H3K4 Me3-EuK mAb conjugate, 500 Assay Points

This Eu-cryptate conjugate allows specific detection of the K4Me3 motif on histone H3, as a result of methylation enzymatic activity.

For research use only. Not for use in diagnostic procedures.
Part Number: 61KA3KAE
Unit Size: 500 Assay Points
Part Number: 61KA3KAD
Unit Size: 10,000 Assay Points
  • Revvity offers a line of ready to use HTRF toolbox reagents for the construction of Histone H3 methyltransferase (HMT), demethylase (HDM) and deacetylase (HDAC) activity assays. This monoclonal antibody Eu-cryptate conjugate is specific for the histone H3K4Me3 methylation mark. It can be used for the development of biochemical assays with the highest levels of specificity and sensitivity, for the screening and study of HMTs, such as MLL1.

  • Assay Points
    500
    Assay Target Type
    Fluorescent reagent
    Assay Technology
    HTRF
    Brand
    HTRF
    Quantity
    1
    Therapeutic Area
    Metabolism/Diabetes
    Neuroscience
    Oncology & Inflammation
    Unit Size
    500 Assay Points
  • Assay principle

    Cisbios HTRF-based reagents give researchers the flexibility to create highly specific, custom histone modification assays for enzymes such as HMTs, DMTs or HDACs. All these assays use a biotinylated histone H3 peptide substrate, a Eu-cryptate labeled detection antibody and XL665-conjugated Streptavidin. Assay specificity is derived from the combination of the chosen Eu-cryptate labeled anti-histone antibody together with the biotinylated peptide substrate. HTRF signal is proportional to the concentration of methyl-specific peptide (e.g. unmethylated, mono-, di- or tri-methylated peptide).

    HTRF Anti histone H3K4 1.svg

     

    Assay protocol

    Two step assays are performed in a single well, require no wash steps and can easily be miniaturized to a 384-well format. The enzymatic reaction containing the biotinylated peptide, enzyme, co-factors and compounds is followed by the detection step in which Eu-labeled anti-methylated histone H3 antibody and SA-XL665 are added. The HTRF signal is detected following a brief room temperature incubation, and is stable for multiple readouts.

    HTRF Anti histone H3K4 2.svg

     

  • Anti-histone H3 Eu-cryptate conjugates

    Methylation level

    K4

    K9

    K27

    k36

    Me0

    V

    V

     

     

    Me1

    V

    V

    V

    V

    Me2

    V

    V

    V

    V

    Me2

    V

     

    V

     

     

    Antibody Specificity

    Each Eu-cryptate labeled anti-histone antibody recognizes a single histone H3 lysine residue and methylation level. Revvity has performed cross-reactivity studies against alternative peptides and methylation levels for each antibody to ensure specificity.

    PeptideMAb H3K4 H3K9 H3K27 H3K36                
      me0 me1 me2 me3 me0 me1 me2 me1 me2 me3 me1 me2
    H3(1-21) 100% 1.3% 0% 0% 100% 0.7% 0% - - 0% - -
    H3(21-44) 0% - - 0% - - 0% 0% 0% 0% 4.4% 0%
    H3(1-21) K4Ac 0% - - - - - - - - - - -
    H3(1-21) K9Ac - - - - 0.1% - - - - - - -
    H3(1-21) K4me1 0% 100% 0.6% 0.5% - - 0% - - 0% - -
    H3(1-21) K4me2  0% 15% 100% 7.4% - - 0% - - 0% - -
    H3(1-21) K4me3 0% 0.4% 0% 100% - - 0% - - 0% - -
    H3(1-21) K9me1 - - - 0% 0.4% 100% 0.1% - - 0% - -
    H3(1-21) K9me2 - - - 0% 0.3% 0% 100% - - 0% - -
    H3(1-21) K9me3 - - - 0.2% 0.1% 0% 0.1% - - 0.2% - -
    H3(21-44) K27me1 - - - 0% - - 0.1% 100% 1.5% 0.2% - 0%
    H3(21-44) K27me2 - - - 0.2% - - 0% 1.4% 100% 3.3% - 0.1%
    H3(21-44) K27me3 - - - 0% - - 0.1% 0% 0% 100% - 0%
    H3(21-44) K36me1 - - - 0% - - 0.1% - - 0% 100% 0.7%
    H3(21-44) K36me2 - - - 0% - - 0.1% - - 0.1% 0.60% 100%
    H3(21-44) K36me3 - - - 0.1% - - 0.1% - - 0.4% 0% 0%

     

  • Validation of enzymatic assays

    Cisbio has developed a number of HTRF assays for HMT/HDM/HDAC enzymes using the antibody conjugate epigenetic panel. The table below recaps the assay set up for H3K4 position, and provides in each case a technical note including materials and methods as well as the results obtained.

    Enzyme Type Methylation level Biotinylated substrate Eu3+-Cryptate MAb  
    LSD1 Demethylase K4 me1 →me0 H3(1-21)K4me1 H3K4me0  
    LSD2 Demethylase K4 me2 →me0 H3(1-21)K4me2 H3K4me0  
    SET7/9 Methyl transferase K4 me0 →me1 H3(1-21)K4me0 H3K4me1  
    JARID1A Demethylase K4 me3 →me2 H3(1-21)K4me3 H3K4me2  
    JARID1C Demethylase K4 me3 →me2 H3(1-21)K4me3 H3K4me2  
    MLL1/WARD Methyl transferase K4 me0 →me2 H3(1-50)K4me0 H3K4me2  
    MLL1/WARD Methyl transferase K4 me0 →me3 H3(1-50)K4me0 H3K4me3  
    HDAC2 Demethylase K4 Ac →unmodified H3(1-21)K4Ac H3K4me0  

     

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Guide
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