EPIgeneous™ Binding Domain Assays

EPIgeneous™ Binding Domain Assay Kits for studying epigenetic readers

This biochemical assay platform enables a no-wash, sensitive, reliable detection of protein/protein interaction between epigenetic binding domains and histone sequences modified by epigenetic enzymes. Three assay kits (A, B and C) have already been validated and fully optimized on a selection of 28 key binding domains. For non-validated reader domains, a fourth kit (Discovery) enables you to profile which of the A, B or C kits is the best assay solution. All kits are based on a GST-tagged Binding Domain /biotin-coupled Histone peptide format, and can be run using the same mix-and-read single plate protocol.  EPIgeneous™ Binding domain assay kits are ideal assay tools for both primary and secondary screening as well as for further inhibitor studies.

Assay principle

EPIgeneous™ Binding Domain assay kits are intended for the simple and rapid assessment of reader domain binding. GST-tagged reader domain protein binding to the biotinylated peptide substrate is detected by a conjugate mix: donor Cryptate labeled antibody raised against the GST tag (donor), and streptavidin-acceptor, for the detection of the biotin-peptide. The interaction of the reader domain with the substrate brings the donor and acceptor dyes into close proximity, and allows FRET to occur upon light excitation. The specific signal at 665 nm is inhibited when a specific compound prevents the reader domain protein from binding to its substrate.

The assay procedure is straightforward, does not require any washing or separation steps, and is carried out without transfer in a single assay plate.

Product Performance

Full assay optimization performed on BRD4(1) using EPIgeneous™ Binding Domain Kit A. Additional data describes the use of EPIgeneous™ Binding Domain Kit A for the profiling of reference (+)-JQ1 compound on multiple binding domains.

1. Peptide-biotin titration - Measurement of BRD4(1) / histone H4 peptide interaction.

The GST-Reader concentration was fixed at 5nMf while the peptide-biotin was serially diluted (100nMf – 0.005nMf). For each peptide-biotin concentration, a negative control was performed by removing the GST-Reader protein from the wells. This negative control was used as non specific signal to calculate the HTRF delta ratio. This specific signal was proportional to the specific interaction measured between GST-BRD4(1) and [Lys(5,8,12,16)Ac]-H4(1-21)-biotin peptide. The 4nM Kd value was determined from this experiment using a one-site specific binding regression (saturation equations).

2. DMSO tolerance - Selection of optimal peptide-biotin concentration depending on DMSO % used.

A

B

A. Peptide-biotin titration performed with various DMSO percentages. The apparent Kd values are determined using one-site specific binding regression (saturation equations). Due to the competitive inhibitor nature of the DMSO on the BRD4(1)/H4 peptide interaction (1), a shift of apparent Kd, is observed while DMSO% increases.

B. As the DMSO competes on BRD4(1)/H4 peptide interaction, the assay window decreases while the DMSO percentage increases. The assay is then recovered by increasing the peptide-biotin concentration. In the case shown here, the optimal peptide-biotin concentration was set between the real Kd and the EC100 obtained for the titration without DMSO, a compromise between assay window and assay sensitivity to enable inhibitor studies. For further investigations, 1% DMSO and 3nM peptide-biotin conditions were selected. Note that the higher the %DMSO and the peptide-biotin concentration, the higher the inhibitor IC50 is.

3. Inhibitor titration - BRD4(1) HTRF inhibition assay using reference inhibitors

The assay was performed using 3 nM peptide-biotin, 5 nM GST-BRD4(1) and 1% DMSO, all set constant throughout the inhibitor titration.

The IC50 of (+)-JQ1 and H4 tetra-acetylated peptide are in good agreement with published data (1, 2).

4. Z’ Factor - Assay robustness

The assay was performed using 3 nM peptide-biotin, 5 nM GST-BRD4(1) and 1% DMSO.

The 0.76 Z’ factor underlines the robustness of the assay and its suitability for HTS.

5. Compound profiling - Compounds selectivity assessment over a selection of validated reader domain assays.

(+)-JQ1 compound was profiled on the BET bromodomain family, CREBBP and BAZ2B bromodomains using EPIgeneous Binding Domain Kit A and B. As already described (3), (+)-JQ1 is a non-selective inhibitor over the BET family but displays selectivity over non BET bromodomains (CREBBP and BAZ2B).

(1)Philpott et al. Mol. BioSyst., 2011, 7, 2899-2908
(2)Filippakopoulos and Knapp, FEBS Letters 586 (2012) 2692–2704
(3)Filippakopoulos et al. Nature 2010

Validated Binding Domain

Three assay kits (A, B and C) have already been validated and fully optimized on a selection of 28 key binding domains. For non-validated reader domains, a fourth one, the Discovery Kit, enables the user to profile which of the A, B or C kits is the best assay solution.

Part#, inserts & MSDS

Ordering Info

DescriptionCat. noProduct insertMSDS
Binding Domain kit A - 500 tests62BDAPEG
Binding Domain kit A - 10,000 tests62BDAPEH
Binding Domain kit B - 500 tests62BDBPEG
Binding Domain kit B - 10,000 tests62BDBPEH
Binding Domain kit C - 500 tests62BDCPEG
Binding Domain kit C - 10,000 tests62BDCPEH
Binding Domain Discovery kit - 500 tests62BDDPEG

Companion products

DescriptionCat. noProduct insertMSDS
Binding Domain Detection buffer #1 - 130 ml62DB1FDG
Binding Domain Detection buffer #2 - 130 ml62DB2FDG
Binding Domain diluent buffer - 200 ml62DLBDDF

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