Journal: ACS Chemical Biology

Article Title: Structure-Guided DOT1L Probe Optimization by Label-Free Ligand Displacement

doi: 10.1021/cb500796d

Figure Lengend Snippet: Development of nonradiometric biochemical and cellular assays for DOT1L. (a–c) AlphaScreen proximity bead-based assay demonstrating adaptability to high-throughput screening ( Z ′ calculated by 1 – ((3σ FED1 + σ DMSO )/absolute value(μ FED1 – μ DMSO )), and expected comparable potency differentiation of known inhibitors. (d–f) Fluorescence polarization assay demonstrating significant assay robustness (Z′ calculated with above formula) and separation of weak DOT1L inhibitors ( SAH ) from more potent compounds ( FED1 and EPZ004777 ). (g–i) High-content imaging assay evaluating H3K79me2 abundance by immunofluorescence in A431 cells after 4 days of indicated DOT1L inhibitors, with diminished H3K79me2 compared to DMSO. Assay is robust (Z′ calculated as above) and reports cellular EC 50 . Biochemical assays were performed in duplicate, and high-content assays were performed as four replicates.

Article Snippet: The addition of 10 uL of 2× this solution to the plates (AlphaScreen plates, PerkinElmer #6005359) was performed with a liquid handler.

Techniques: Amplified Luminescent Proximity Homogenous Assay, Bead-based Assay, High Throughput Screening Assay, Fluorescence, Imaging, Immunofluorescence

Journal: Bioorganic & medicinal chemistry

Article Title: Identification of the first small-molecule inhibitor of the REV7 DNA repair protein interaction

doi: 10.1016/j.bmc.2016.07.026

Figure Lengend Snippet: A) Dose response of indicated biotin-tagged peptide titrated over His-REV7(R124A)/REV3L(1847–1898) in AlphaScreen. A hooking peak characteristic to AlphaScreen assay is observed only for REV3L(1875–1895)-biotin peptide. B) Dose response of competition by the indicated peptide titrated over a mixture of His-REV7(R124A)/REV3L(1847–1898) and REV3L(1875–1895)-biotin peptide. IC50 for REV3L(1875–1895) is read as 80 nM. An unrelated peptide of similar length [Fz7(544–566)] served as a negative control. Error bars represent standard deviation (n = 2). Hit identification and validation by independent assays. C) Structure of the hit compound 1. D) Destabilization, but not nonspecific denaturation, of His-REV7(R124A)/REV3L(1847–1898) protein by compound 1 measured by thermal shift assay. E) Dissociation of His-REV7(R124A)/biotin-AviTag-REV3L(1847–1898) protein complex (1 μM) by compound 1 (50 μM) or REV3L(1875–1895) peptide (5 μM), on streptavidin-conjugated agarose beads. DMSO was used as a vehicle control for compound 1. Each treatment was duplicated, as shown by the bars.

Article Snippet: After incubating the plates overnight at room temperature, AlphaScreen nickel chelate (Ni-NTA) acceptor beads (PerkinElmer) diluted 1:100 in 10 μL of the AlphaScreen buffer were added by using the Wellmate liquid dispenser.

Techniques: Amplified Luminescent Proximity Homogenous Assay, Negative Control, Standard Deviation, Thermal Shift Assay

Journal: Journal of biomolecular screening

Article Title: A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors.

doi: 10.1177/108705710200700102

Figure Lengend Snippet: FIG. 1. Schematic diagram of FXR assay formats. The bile acid CDCA is needed to induce a complex between the FXR–GST and the coactivator-derived peptide SRC-1. (A) TR-FRET format, where an energy transfer between europium chelate and allophycocyanin (APC) occurs. (B) ALPHAScreen format, where the excitation of a donor bead at 680 nm produces singlet oxygen, thus diffusing to an ac- ceptor bead and undergoing a chemiluminescent reaction. (C) TRF-based plate-binding assay. A binding reaction occurs in a plate coated with NeutrAvidin. The complex is captured via a biotin–NeutrAvidin interaction, the plate is washed to separate unbound reagents, and GST is detected with a europium chelate–labeled antibody, the europium being released and detected by the addition of an enhancement solution.

Article Snippet: The 1536-well assays were performed in 8-ml volumes in Greiner (Greiner America, Inc., Lake Mary, FL) black polystyrene plates for TR-FRET (model 782072) and Greiner white polystyrene plates for ALPHAScreen (model 782075).

Techniques: Derivative Assay, Amplified Luminescent Proximity Homogenous Assay, Binding Assay, Labeling

Journal: Journal of biomolecular screening

Article Title: A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors.

doi: 10.1177/108705710200700102

Figure Lengend Snippet: FIG. 2. Time course of binding reaction. All assays (triplicate) were performed at optimal concentration of detector reagents (see Table 1). Aliquots of 50 mM CDCA were added at time zero to a reaction buffer containing 10 nM SRC1–biotin and 1 nM FXR–GST for TR-FRET and ALPHAScreen (AS). For TRF, the plates were coated with 100 nM SRC1–biotin and the [FXR–GST] was 0.5 nM. (A) TR-FRET assay. (B) ALPHAScreen assay. (C) TRF plate-binding assay.

Article Snippet: The 1536-well assays were performed in 8-ml volumes in Greiner (Greiner America, Inc., Lake Mary, FL) black polystyrene plates for TR-FRET (model 782072) and Greiner white polystyrene plates for ALPHAScreen (model 782075).

Techniques: Binding Assay, Concentration Assay, Amplified Luminescent Proximity Homogenous Assay

Journal: Journal of biomolecular screening

Article Title: A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors.

doi: 10.1177/108705710200700102

Figure Lengend Snippet: FIG. 3. SRC1–biotin and FXR–GST dose–response curves. Qua- druplicate assays in 30-ml volumes were measured in 384-well plates containing 50 mM CDCA, varied concentrations of bi- otin–SRC1, and varied concentrations of FXR–GST. The detector reagents were fixed as shown in Table 1 and the binding reaction time was 2.5 h. (A) TR-FRET assay. (B) ALPHAScreen assay. (C) TRF plate-binding assay.

Article Snippet: The 1536-well assays were performed in 8-ml volumes in Greiner (Greiner America, Inc., Lake Mary, FL) black polystyrene plates for TR-FRET (model 782072) and Greiner white polystyrene plates for ALPHAScreen (model 782075).

Techniques: Binding Assay, Amplified Luminescent Proximity Homogenous Assay

Journal: Journal of biomolecular screening

Article Title: A comparison of ALPHAScreen, TR-FRET, and TRF as assay methods for FXR nuclear receptors.

doi: 10.1177/108705710200700102

Figure Lengend Snippet: FIG. 4. CDCA dose–response curves for assays in 1536-well Greiner plates. (A) TR-FRET format: quadruplicate assays in 8-ml vol- umes contained 25 nM biotin–SRC1 and 2.5 nM FXR–GST. Kd (app) 5 13 mM. (B) ALPHAScreen format: quadruplicate assays in 8- ml volumes contained 25 nM biotin–SRC1 and 2.5 nM FXR–GST. Kd (app) 5 21 mM. (C) TRF plate-binding assay in 40-ml volumes (384-well format). [FXR–GST] 5 0.5 nM. Plate coated with 100 nM SRC1–biotin. Kd (app) 5 30–47 mM.

Article Snippet: The 1536-well assays were performed in 8-ml volumes in Greiner (Greiner America, Inc., Lake Mary, FL) black polystyrene plates for TR-FRET (model 782072) and Greiner white polystyrene plates for ALPHAScreen (model 782075).

Techniques: Amplified Luminescent Proximity Homogenous Assay, Binding Assay