Binding Assay:Article Title: Cyclic dinucleotide compounds
Article Snippet: .. The binding of compounds of the invention to human STING may be demonstrated using the following assay: Differential Scanning Fluorimetry (DSF) Materials: Hard-Shell®PCR Plates 384-Well thin-wall (Catalog# HSP3805R, BIO-RAD) Microseal®‘B’ Adhesive Seals for PCR Plates (Catalog# MSB-1001, BIO-RAD) SYPRO orange solution in DMSO (SIGMA cat.-no. S5692-500UL), concentration “5000×” Instrumentation: Reader: CFX384 Real-Time System (Bio-Rad) Pipetting Robot: HamiltonStarlet Assay buffer: 20 mM Tris, 150 mM NaCl pH7.5 Target Protein: Human STING (hSTING, residues 155-341, wild-type sequence with N-terminal His8-tag and TEV-cleavage site, MW: 23601.5 Da) Protein stock solution: c=309 μM stock solution in assay buffer Final Assay concentrations of test compounds: 100 uM, 3 uM target protein, “5×” SYPR Orange Assay Procedure: 1) Compound stock solutions and dilutions thereof were prepared in assay buffer 2) 5 ul fluorescent dye stock solution (5000× SYPRO Orange) was mixed with 50 ul target protein (309 uM) and 945 ul buffer. ..
Adhesive:Article Title: Cyclic dinucleotide compounds
Article Snippet: .. The binding of compounds of the invention to human STING may be demonstrated using the following assay: Differential Scanning Fluorimetry (DSF) Materials: Hard-Shell®PCR Plates 384-Well thin-wall (Catalog# HSP3805R, BIO-RAD) Microseal®‘B’ Adhesive Seals for PCR Plates (Catalog# MSB-1001, BIO-RAD) SYPRO orange solution in DMSO (SIGMA cat.-no. S5692-500UL), concentration “5000×” Instrumentation: Reader: CFX384 Real-Time System (Bio-Rad) Pipetting Robot: HamiltonStarlet Assay buffer: 20 mM Tris, 150 mM NaCl pH7.5 Target Protein: Human STING (hSTING, residues 155-341, wild-type sequence with N-terminal His8-tag and TEV-cleavage site, MW: 23601.5 Da) Protein stock solution: c=309 μM stock solution in assay buffer Final Assay concentrations of test compounds: 100 uM, 3 uM target protein, “5×” SYPR Orange Assay Procedure: 1) Compound stock solutions and dilutions thereof were prepared in assay buffer 2) 5 ul fluorescent dye stock solution (5000× SYPRO Orange) was mixed with 50 ul target protein (309 uM) and 945 ul buffer. ..
Polymerase Chain Reaction:Article Title: Cyclic dinucleotide compounds
Article Snippet: .. The binding of compounds of the invention to human STING may be demonstrated using the following assay: Differential Scanning Fluorimetry (DSF) Materials: Hard-Shell®PCR Plates 384-Well thin-wall (Catalog# HSP3805R, BIO-RAD) Microseal®‘B’ Adhesive Seals for PCR Plates (Catalog# MSB-1001, BIO-RAD) SYPRO orange solution in DMSO (SIGMA cat.-no. S5692-500UL), concentration “5000×” Instrumentation: Reader: CFX384 Real-Time System (Bio-Rad) Pipetting Robot: HamiltonStarlet Assay buffer: 20 mM Tris, 150 mM NaCl pH7.5 Target Protein: Human STING (hSTING, residues 155-341, wild-type sequence with N-terminal His8-tag and TEV-cleavage site, MW: 23601.5 Da) Protein stock solution: c=309 μM stock solution in assay buffer Final Assay concentrations of test compounds: 100 uM, 3 uM target protein, “5×” SYPR Orange Assay Procedure: 1) Compound stock solutions and dilutions thereof were prepared in assay buffer 2) 5 ul fluorescent dye stock solution (5000× SYPRO Orange) was mixed with 50 ul target protein (309 uM) and 945 ul buffer. ..
Concentration Assay:Article Title: Cyclic dinucleotide compounds
Article Snippet: .. The binding of compounds of the invention to human STING may be demonstrated using the following assay: Differential Scanning Fluorimetry (DSF) Materials: Hard-Shell®PCR Plates 384-Well thin-wall (Catalog# HSP3805R, BIO-RAD) Microseal®‘B’ Adhesive Seals for PCR Plates (Catalog# MSB-1001, BIO-RAD) SYPRO orange solution in DMSO (SIGMA cat.-no. S5692-500UL), concentration “5000×” Instrumentation: Reader: CFX384 Real-Time System (Bio-Rad) Pipetting Robot: HamiltonStarlet Assay buffer: 20 mM Tris, 150 mM NaCl pH7.5 Target Protein: Human STING (hSTING, residues 155-341, wild-type sequence with N-terminal His8-tag and TEV-cleavage site, MW: 23601.5 Da) Protein stock solution: c=309 μM stock solution in assay buffer Final Assay concentrations of test compounds: 100 uM, 3 uM target protein, “5×” SYPR Orange Assay Procedure: 1) Compound stock solutions and dilutions thereof were prepared in assay buffer 2) 5 ul fluorescent dye stock solution (5000× SYPRO Orange) was mixed with 50 ul target protein (309 uM) and 945 ul buffer. ..
Sequencing:Article Title: Cyclic dinucleotide compounds
Article Snippet: .. The binding of compounds of the invention to human STING may be demonstrated using the following assay: Differential Scanning Fluorimetry (DSF) Materials: Hard-Shell®PCR Plates 384-Well thin-wall (Catalog# HSP3805R, BIO-RAD) Microseal®‘B’ Adhesive Seals for PCR Plates (Catalog# MSB-1001, BIO-RAD) SYPRO orange solution in DMSO (SIGMA cat.-no. S5692-500UL), concentration “5000×” Instrumentation: Reader: CFX384 Real-Time System (Bio-Rad) Pipetting Robot: HamiltonStarlet Assay buffer: 20 mM Tris, 150 mM NaCl pH7.5 Target Protein: Human STING (hSTING, residues 155-341, wild-type sequence with N-terminal His8-tag and TEV-cleavage site, MW: 23601.5 Da) Protein stock solution: c=309 μM stock solution in assay buffer Final Assay concentrations of test compounds: 100 uM, 3 uM target protein, “5×” SYPR Orange Assay Procedure: 1) Compound stock solutions and dilutions thereof were prepared in assay buffer 2) 5 ul fluorescent dye stock solution (5000× SYPRO Orange) was mixed with 50 ul target protein (309 uM) and 945 ul buffer. ..
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