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alphascreen fluorescence signal biotin  (Mimetics)
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Mimetics alphascreen fluorescence signal biotin
Development of a high-throughput <t>AlphaScreen</t> for candidate inhibitors of the MIF promoter microsatellite - ICBP90 interaction. A , AlphaScreen analyses of the dose-dependent solution interaction of the 5′-biotin MIF promoter CATT 8 oligonucleotide (5′Biotin-CATT 8 , 6.8–5000 nM) with ICBP90 413-618 at the tested concentrations of 6.8, 21.0, and 62.0 nM. <t>Fluorescence</t> intensity is expressed in counts per second (cps). B , dose-dependent inhibition of anti-ICPB90 IgG or an isotypic control IgG antibody on AlphaScreen solution interaction of ICBP90 413-618 (62 nM) with 5′biotin MIF promoter CATT 8 oligonucleotide (10 nM). C , electrophoretic mobility shift assay (EMSA) of five representative compound hits identified by AlphaScreen (Compound 1: NSC 3852, 2: NSC 5159, 3: NSC 106995 (CMFT), 4: NSC 114341, and 5: NSC 11107). The lanes show retention of ICBP90 413-618 /5′Biotin-CATT 8 molecular complexes, dependence on ICBP90 413-618 , and inhibition by compound 3; Lane 1: 5′Biotin MIF promoter CATT 8 only; Lane 2: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 ; Lanes 3 to 7: 5′Biotin MIF promoter CATT 8 + ICBP90 413-618 plus test Compounds 1 to 5; Lanes 8 to 12: 5′Biotin MIF promoter CATT 8 plus test compounds 1 to 5 without ICBP90 413-618 . D , dose-dependent inhibition of ICBP90 413-618 /5′Biotin-CATT 8 solution interaction by CMFT (NSC106995) and 3 structural congeners identified in the screened libraries (NSC 1569419, 382951, 382953). E , dose-dependent inhibition by CMFT of ICBP90 413-618 /5′Biotin-CATT 8 complexation assessed by EMSA. Lane 1: 5′Biotin MIF promoter CATT 8 only; Lane 2: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 ; Lane 3: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 plus excess 5′ MIF promoter CATT 8 oligonucleotide. Lanes 4 to 11: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 plus increasing concentrations of CMFT (0, 7.8, 15.6, 31.2, 62.5, 125, 250, and 500 μM). Graphed data points are duplicate or triplicate determinations with mean ± SD (Student’s t test, two-tailed). EMSA determinations are representative of at least two independent experiments.
Alphascreen Fluorescence Signal Biotin, supplied by Mimetics, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Development of a high-throughput AlphaScreen for candidate inhibitors of the MIF promoter microsatellite - ICBP90 interaction. A , AlphaScreen analyses of the dose-dependent solution interaction of the 5′-biotin MIF promoter CATT 8 oligonucleotide (5′Biotin-CATT 8 , 6.8–5000 nM) with ICBP90 413-618 at the tested concentrations of 6.8, 21.0, and 62.0 nM. Fluorescence intensity is expressed in counts per second (cps). B , dose-dependent inhibition of anti-ICPB90 IgG or an isotypic control IgG antibody on AlphaScreen solution interaction of ICBP90 413-618 (62 nM) with 5′biotin MIF promoter CATT 8 oligonucleotide (10 nM). C , electrophoretic mobility shift assay (EMSA) of five representative compound hits identified by AlphaScreen (Compound 1: NSC 3852, 2: NSC 5159, 3: NSC 106995 (CMFT), 4: NSC 114341, and 5: NSC 11107). The lanes show retention of ICBP90 413-618 /5′Biotin-CATT 8 molecular complexes, dependence on ICBP90 413-618 , and inhibition by compound 3; Lane 1: 5′Biotin MIF promoter CATT 8 only; Lane 2: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 ; Lanes 3 to 7: 5′Biotin MIF promoter CATT 8 + ICBP90 413-618 plus test Compounds 1 to 5; Lanes 8 to 12: 5′Biotin MIF promoter CATT 8 plus test compounds 1 to 5 without ICBP90 413-618 . D , dose-dependent inhibition of ICBP90 413-618 /5′Biotin-CATT 8 solution interaction by CMFT (NSC106995) and 3 structural congeners identified in the screened libraries (NSC 1569419, 382951, 382953). E , dose-dependent inhibition by CMFT of ICBP90 413-618 /5′Biotin-CATT 8 complexation assessed by EMSA. Lane 1: 5′Biotin MIF promoter CATT 8 only; Lane 2: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 ; Lane 3: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 plus excess 5′ MIF promoter CATT 8 oligonucleotide. Lanes 4 to 11: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 plus increasing concentrations of CMFT (0, 7.8, 15.6, 31.2, 62.5, 125, 250, and 500 μM). Graphed data points are duplicate or triplicate determinations with mean ± SD (Student’s t test, two-tailed). EMSA determinations are representative of at least two independent experiments.

Journal: The Journal of Biological Chemistry

Article Title: A small-molecule allele-selective transcriptional inhibitor of the MIF immune susceptibility locus

doi: 10.1016/j.jbc.2024.107443

Figure Lengend Snippet: Development of a high-throughput AlphaScreen for candidate inhibitors of the MIF promoter microsatellite - ICBP90 interaction. A , AlphaScreen analyses of the dose-dependent solution interaction of the 5′-biotin MIF promoter CATT 8 oligonucleotide (5′Biotin-CATT 8 , 6.8–5000 nM) with ICBP90 413-618 at the tested concentrations of 6.8, 21.0, and 62.0 nM. Fluorescence intensity is expressed in counts per second (cps). B , dose-dependent inhibition of anti-ICPB90 IgG or an isotypic control IgG antibody on AlphaScreen solution interaction of ICBP90 413-618 (62 nM) with 5′biotin MIF promoter CATT 8 oligonucleotide (10 nM). C , electrophoretic mobility shift assay (EMSA) of five representative compound hits identified by AlphaScreen (Compound 1: NSC 3852, 2: NSC 5159, 3: NSC 106995 (CMFT), 4: NSC 114341, and 5: NSC 11107). The lanes show retention of ICBP90 413-618 /5′Biotin-CATT 8 molecular complexes, dependence on ICBP90 413-618 , and inhibition by compound 3; Lane 1: 5′Biotin MIF promoter CATT 8 only; Lane 2: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 ; Lanes 3 to 7: 5′Biotin MIF promoter CATT 8 + ICBP90 413-618 plus test Compounds 1 to 5; Lanes 8 to 12: 5′Biotin MIF promoter CATT 8 plus test compounds 1 to 5 without ICBP90 413-618 . D , dose-dependent inhibition of ICBP90 413-618 /5′Biotin-CATT 8 solution interaction by CMFT (NSC106995) and 3 structural congeners identified in the screened libraries (NSC 1569419, 382951, 382953). E , dose-dependent inhibition by CMFT of ICBP90 413-618 /5′Biotin-CATT 8 complexation assessed by EMSA. Lane 1: 5′Biotin MIF promoter CATT 8 only; Lane 2: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 ; Lane 3: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 plus excess 5′ MIF promoter CATT 8 oligonucleotide. Lanes 4 to 11: 5′Biotin MIF promoter CATT 8 plus ICBP90 413-618 plus increasing concentrations of CMFT (0, 7.8, 15.6, 31.2, 62.5, 125, 250, and 500 μM). Graphed data points are duplicate or triplicate determinations with mean ± SD (Student’s t test, two-tailed). EMSA determinations are representative of at least two independent experiments.

Article Snippet: Positively scoring compounds were further tested for interference with the AlphaScreen fluorescence signal ( e.g. , biotin mimetics) using the AphaScreen PerkinElmer TrueHitsKit (#6760627D).

Techniques: High Throughput Screening Assay, Amplified Luminescent Proximity Homogenous Assay, Fluorescence, Inhibition, Control, Electrophoretic Mobility Shift Assay, Two Tailed Test