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November 15, 2010 (Vol. 30, No. 20)

Epigenetic Modifications on Histones

Homogeneous Nonradioactive Assays to Monitor Methylation Process

  • Click Image To Enlarge +
    Figure 2. AlphaLISA SET7/9 methyltransferase assay optimization: All enzymatic reactions were performed in 10 µL (final assay volume: 25 µL). Detection has been carried out with Acceptor beads conjugated to an antibody specific against the mono- and di-methylated forms of the Histone H3 peptide substrate (H3K4me1-2). (A) Enzymatic time course, at 50 nM biotin-H3 peptide substrate, and 100 µM SAM (saturating concentration). (B) SAM dose-response curve (1 nM SET7/9, 50 nM biotin-H3 peptide substrate). (C) Inhibitor dose-response curves (1 nM SET7/9, 50 nM biotin-H3 peptide substrate, 100 nM SAM).

    Determination of optimal enzymatic assay conditions has to be performed for each enzyme studied. Figures 1 and 2 provide examples of results obtained following the successful optimization of a SET7/9 assay (H3 Lys4 methylation) using either Lance Ultra or AlphaLISA detection platforms. With both assays, signal increase was proportional to enzyme reaction time and enzyme concentration (Figure 1A and 2A). Apparent Km values of 75 and 79 nM were obtained for the SAM co-substrate in Lance Ultra and AlphaLISA assays, respectively (Figure 1B and 2B).

    In addition, rank order of potency for two known methyltransferase inhibitors (SAH, sinefungin) was as expected and further validated the assay suitability for studying small molecule inhibitors (Figure 1C and 2C). Low intra-assay variability was confirmed by Z´-factor values over 0.8, generated with both technologies in a study using maximal inhibition with sinefungin (data not shown).

  • Conclusions

    Click Image To Enlarge +
    Figure 3. Lance Ultra and AlphaLISA platforms in the detection of histone modifications: Detection of epigenetic modifications on a biotinylated peptide substrate can be accomplished using either the Lance Ultra (TR-FRET) or AlphaLISA (proximity-based chemiluminescent) platforms. In Lance Ultra, excitation at 320–340 nm of a Europium chelate conjugated antimark antibody enables the resonance energy transfer to the acceptor ULight dye conjugated to streptavidin. This results in the emission of photons at 665 nm. In AlphaLISA, excitation of the streptavidin Donor beads at 680 nm triggers the release of short-lived singlet oxygen molecules that activate the Acceptor beads in close proximity to emit light at 615 nm. In both cases, the signal output is proportional to the amount of modified biotin-peptide product.

    As exemplified in this SET7/9 study, peptide substrate and enzyme concentrations in the nanomolar range were sufficient to generate robust and reproducible assay signals using either Lance Ultra or AlphaLISA reagents. Figure 3 shows a scheme of how the signal is produced on both platforms. This represents a significant advantage over other technologies using colorimetric or fluorescence-based detection methods that generally require micromolar substrate concentrations.

    More significantly, our Lance Ultra and AlphaLISA reagents allowed the use of mid-nanomolar concentrations of SAM, facilitating the screening of SAM competitive inhibitors.

    The Lance Ultra europium-antibodies and AlphaLISA Acceptor beads developed for epigenetic research are available as stand-alone reagents, allowing the user maximum flexibility for assay development with preferred substrate and enzyme source. Due to the simple all-in-one-well format, the small number of steps involved (usually 3 or 4), and the absence of wash steps, Lance Ultra and AlphaLISA assays for epigenetic research are amenable to automation. For all these reasons, they constitute a good choice not only for HTS campaigns (hit finding) but also for studies involving secondary screenings and orthogonal testing (hit-to-lead studies).

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