Adp glo reagent

Manufactured by Promega

Sourced in United States, United Kingdom

The ADP-Glo reagent is a laboratory product designed to detect and quantify the presence of ADP (Adenosine Diphosphate) in a sample. It functions by converting ADP into ATP (Adenosine Triphosphate), which is then measured using a luminescent assay. The core purpose of the ADP-Glo reagent is to provide a sensitive and reliable method for the analysis of ADP levels in various experimental settings.

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Lab products found in correlation

Kinase detection reagent, by Promega (16 mentions) Adp glo kinase assay kit, by Promega (4 mentions) Prism 5, by GraphPad (3 mentions) Adenosine triphosphate (atp), by Promega (2 mentions) Adp glo kinase assay, by Promega (2 mentions) Pin transfer robot, by Seiko Instruments (2 mentions) Synergy 4, by Agilent Technologies (2 mentions) 1 2 dioleoyl sn glycerol, by Merck Group (2 mentions) Phosphatidylserine, by Merck Group (2 mentions) Infinite m1000 microplate reader, by Tecan (2 mentions) 384 well plate, by PerkinElmer (2 mentions) Akt substrate 2 peptide ckrpraasfae, by Promega (2 mentions) Nivo s luminometer, by PerkinElmer (2 mentions) Xfluor software, by Tecan (1 mentions) Adenosine diphosphate (adp), by Promega (1 mentions) Adp glo, by Promega (1 mentions) Half area 96 well microplates, by Corning (1 mentions) M200 pro, by Tecan (1 mentions) Fluostar omega, by BMG Labtech (1 mentions) White 384 well microplates, by Greiner (1 mentions)

Spelling variants of this product

ADP-Glo (25 citations) ADP-GloTM reagent (3 citations) ADP-Glo reagent (ADP-Glo kinase assay kit; (3 citations)

37 protocols using adp glo reagent

Kinetic Analyses of FLT3-ITD Inhibition

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Kinetic analyses of FLT3-ITD were performed using a luminometric kinase assay varying the concentration of ATP using the ADP-Glo reagents (Promega). The serially diluted A674563 and FLT3-ITD (40 ng/μL) were assayed in a reaction (10 μL) containing 40 mM Tris (pH 7.6), 20 mM MgCl₂, 2 mM MnCl₂, 50 μM DTT. After 60 min incubation at RT, various concentrations of ATP, 0.2 μg/μL FLT3 substrate Poly (4:1 Glu, Tyr) peptide added and incubated for 60 min at 37°C. The overall rate of reaction is determined as the slope of the decreasing phase of the reaction. Each data point was collected in duplicate and kinetic parameters were obtained using Prism 5.0 (GraphPad Software, San Diego, CA).

Wang A., Wu H., Chen C., Hu C., Qi Z., Wang W., Yu K., Liu X., Zou F., Zhao Z., Wu J., Liu J., Liu F., Wang L., Stone R.M., Galinksy I.A., Griffin J.D., Zhang S., Weisberg E.L., Liu J, & Liu Q. (2016). Dual inhibition of AKT/FLT3-ITD by A674563 overcomes FLT3 ligand-induced drug resistance in FLT3-ITD positive AML. Oncotarget, 7(20), 29131-29142.

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Recombinant human ALK kinase assay

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N-terminal 6His tagged recombinant human ALK expressed in baculovirus Sf21 was purchased from EMD Millipore (Billerica, MA, USA) (purity ≥60% by sodium dodecyl sulfate polyacrylamide gel electrophoresis) and aliquoted to 1 μL fractions when it was used the first time (to avoid multiple freeze/thaws for subsequent experiments). BNP7787 was prepared by a proprietary method (purity >97%, no mesna was detected by mass spectroscopy). Kinase inhibitor, PF02341066 (crizotinib), was purchased from Selleck Chemicals, LLC. (Houston TX, USA). Polyglutamate-tyrosine (PolyGT) substrate was purchased from Sigma-Aldrich (St. Louis, MO, USA). Kinase assay buffer was prepared and consisted of 20 mM HEPES, 0.1% Brij 96, 10 mM NaF, 1 mM Na₃VO₄, and 10 mM MnCl₂ adjusted to a final pH of 7.5. Half-area 96-well microplates were purchased directly from Corning Incorporated (Corning, NY, USA). ADP-Glo reagents were purchased from Promega (Madison WI, USA) and consisted of ADP, ATP, ADP-Glo, kinase detection reagent buffer, and kinase detection substrate. All other reagents were purchased from Sigma-Aldrich Co (St Louis, MO, USA). A Tecan Ultra microplate reader with XFluor software (V4.51; Tecan [Morrisville, NC, USA]) and RdrOle software (V4.50; Tecan) were used in this study.

Parker A.R., Petluru P.N., Nienaber V.L., Zhao M., Ayala P.Y., Badger J., Chie-Leon B., Sridhar V., Logan C., Kochat H, & Hausheer F.H. (2015). Novel covalent modification of human anaplastic lymphoma kinase (ALK) and potentiation of crizotinib-mediated inhibition of ALK activity by BNP7787. OncoTargets and therapy, 8, 375-383.

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Affinity Purification and Kinase Assay

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SIRK1-GFP and QSK1-GFP fusion proteins were affinity purified over anti-GFP beads (see above). A luciferase-based kinase activity assay was performed as described (36 (link)). The agarose beads with GFP-tagged proteins were re-suspended into 30 μl kinase reaction buffer with ATP and the generic kinase substrate myelin basic protein (40 mm Tris/HCl pH 7.5, 10 mm MgCl₂, 0.1% BSA, 2 mm DTT, 100 μm ATP, 0.4 μg/μl myelin basic protein). After incubation for one hour, 30 μl ADP-GLO Reagents (Promega, Mannheim, Germany) was added and incubated for 40 min. Then Kinase Detection Reagents were added and incubated for another hour. Luminescence as a measure of ATP conversion from ADP was recorded with a luminometer (Tecan M200 Pro, Crailsheim, Germany). In general, protein isolations from three independent batches of roots were tested, and the average activity value is presented.

Wu X.N., Chu L., Xi L., Pertl-Obermeyer H., Li Z., Sklodowski K., Sanchez-Rodriguez C., Obermeyer G, & Schulze W.X. (2019). Sucrose-induced Receptor Kinase 1 is Modulated by an Interacting Kinase with Short Extracellular Domain. Molecular & Cellular Proteomics : MCP, 18(8), 1556-1571.

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Kinetic Analyses of PI3Kδ and Vps34

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Kinetic analyses of PI3Kδ and Vps34 were performed using a luminometric kinase assay varying the concentration of ATP using the ADP-Glo reagents (Promega). The serially diluted PI3KD/V-IN-01 and PI3Kδ (1.0 μg/mL) were assayed in a reaction (10 μL) containing 50 mM Hepes pH 7.5, 3 mM MgCl₂, 1 mM EGTA, 1 mM NaCl, 0.03% CHAPS, 2 mM DTT, and Vps34 (1.2 μg/mL). In the case of Vps34, 2 mM MnCl2 and 2 mM DTT were further added to the reaction buffer containing 50 mM Hepes pH 7.5, 4 mM MgCl2, 1 mM EGTA and 0.1% CHAPS. After 60 min incubation at RT, varied concentrations of ATP and 0.1 mM substrate (PIP2:PS for PI3Kδ, PI:PS for Vps34) were added and incubated for 60 min at 37° C. The overall rate of reaction was determined as the slope of the decreasing phase of the reaction. Each data point was collected in duplicate and kinetic parameters were obtained using Prism 5.0 (GraphPad Software, San Diego, CA).

Liu X., Wang A., Liang X., Liu J., Zou F., Chen C., Zhao Z., Deng Y., Wu H., Qi Z., Wang B., Wang L., Liu F., Xu Y., Wang W., Fernandes S.M., Stone R.M., Galinsky I.A., Brown J.R., Loh T., Griffin J.D., Zhang S., Weisberg E.L., Zhang X., Liu J, & Liu Q. (2016). Simultaneous inhibition of Vps34 kinase would enhance PI3Kδ inhibitor cytotoxicity in the B-cell malignancies. Oncotarget, 7(33), 53515-53525.

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Kinetic Analysis of PI3KCD Inhibition

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Kinetic analyses of PI3KCD were performed using a luminometric kinase assay varying the concentration of ATP using the ADP-Glo reagents (Promega). The serially diluted PI3KD-IN-015 and PI3KCD (2.2 μg/mL) were assayed in a reaction (10 μL) containing 50 mM HEPES (pH 7.5), 3 mM MgCl₂, 1 mM EGTA, 100 mM NaCl₂, 0.03% CHAPS. After 60 min incubation at RT, varied concentrations of ATP, 0.1mM substrate PIP2:PS were added and incubated for 60 min at 37°C. The overall rate of reaction was determined as the slope of the decreasing phase of the reaction. Each data point was collected in duplicate and kinetic parameters were obtained using Prism 5.0 (GraphPad Software, San Diego, CA).

Liu X., Wang A., Liang X., Chen C., Liu J., Zhao Z., Wu H., Deng Y., Wang L., Wang B., Wu J., Liu F., Fernandes S.M., Adamia S., Stone R.M., Galinsky I.A., Brown J.R., Griffin J.D., Zhang S., Loh T., Zhang X., Wang W., Weisberg E.L., Liu J, & Liu Q. (2016). Characterization of selective and potent PI3Kδ inhibitor (PI3KD-IN-015) for B-Cell malignances. Oncotarget, 7(22), 32641-32651.

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Kinase Inhibition Assay with CLK1

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Recombinant human CLK1 (50 ng, C57-11G, SignalChem Biotech Inc. Canada) was incubated with DMSO or a range of serial dilutions of 1C8 in kinase buffer (40 mM Tris–HCl pH 7.5, 25 mM MgCl₂, 0.1 mg/mL BSA and 0.25 mM DTT) containing 50 µM of ATP (Promega, Madison, WI, USA). The 5 µL kinase reaction performed in 384-wells Bio-Rad white plate was incubated for 30 min at 30 °C. The reactions were stopped by adding 5 μL of ADP-Glo reagent (Promega, V6930) and incubated at room temperature for 40 min. After addition of 10 μL of Kinase Detection Reagent (Promega, V6930), plates were incubated for another 30 min at room temperature. Luminescence was measured with a plate-reading luminometer (integration time 750 ms/well). Data were analysed using GraphPad Prism version 8.3.0 (GraphPad Software, San Diego, California, USA).

Dahal S., Clayton K., Been T., Fernet-Brochu R., Ocando A.V., Balachandran A., Poirier M., Maldonado R.K., Shkreta L., Boligan K.F., Guvenc F., Rahman F., Branch D., Bell B., Chabot B., Gray-Owen S.D., Parent L.J, & Cochrane A. (2022). Opposing roles of CLK SR kinases in controlling HIV-1 gene expression and latency. Retrovirology, 19, 18.

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Optimal Reaction Time for PERK Kinase Assay

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To determine the optimal reaction time, 87 nM of PERK reacted with 50 μM of ATP and 0.26 μM of SMAD3, in presence of 0.87 μM of GSK2606414 (inhibition curve) or calcineurin-B (activation curve), or kinase buffer A (basal curve). Similar to previous experiments, all solutions were prepared in kinase buffer A, at a final reaction volume of 5 μL, and run in white 384-well microplates (Greiner), in triplicates. The kinase reaction was terminated at different time-points—15, 30, 60, and 120 min—by adding 5 μL of ADP-Glo^™ reagent (Promega) and incubating for 40 min at RT. The ATP consumption was measured on a FLUOstar^® Omega plate reader (BMG LabTech), after a 40 min incubation with 10 μL of kinase detection reagent (Promega), at RT, protected from light. After correcting the background as before, data was normalized to PERK basal activity (i.e., condition where neither GSK2606414 nor calcineurin-B were added), in order to calculate the percentage of relative PERK activity. The GSK2606414 (inhibition) and calcineurin-B (activation) curves were obtained by plotting the percentage of relative PERK activity against the respective time-point.

Costa M.F., Höglinger G.U, & Rösler T.W. (2023). Development of a cell-free screening assay for the identification of direct PERK activators. PLOS ONE, 18(5), e0283943.

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Optical Biosensor-based Enzyme Kinetics

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Biacore T200 and 4000 optical biosensors and NTA sensor chips were obtained from GE Healthcare (Little Chalfont, Buckinghamshire, UK).
ADP‐Glo™ Kinase Assay kit which contains ADP‐Glo™ Reagent, kinase detection reagent, ATP, and ADP was purchased from Promega (Madison, WI, USA).
Biodesy Delta 384‐well plates and the SHG‐active dye, SHG2‐maleimide (thiol reactive), were made available by Biodesy, Inc.

Hantani Y., Iio K., Hantani R., Umetani K., Sato T., Young T., Connell K., Kintz S, & Salafsky J. (2018). Identification of inactive conformation‐selective interleukin‐2‐inducible T‐cell kinase (ITK) inhibitors based on second‐harmonic generation. FEBS Open Bio, 8(9), 1412-1423.

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RIPK2 Kinase Inhibition Assay

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Recombinant RIPK2 protein (20 ng per reaction) is diluted in the reaction buffer consisting of 40 mM Tris (pH 7.5); 20 mM MgCl₂; 0.1 mg/ml BSA; 50 μM DTT. Diluted protein is added to low volume white 384 well plates (2 μL/well). Inhibitors are diluted in reaction buffer (final 25% DMSO), 1 μL is added to each well and incubated 5 min at room temperature. Reactions are initiated by the addition of 2 μL of 100 μM ATP and 1 mg/ml RS repeat peptide (SignalChem) in the reaction buffer. Plates are sealed with plastic coverslips and incubated at room temperature for 2 h. Reactions are stopped by the addition of 5 μL of ADPGlo reagent (Promega) and ADP generation reaction is performed for 40 min at room temperature. Luminescence signal is generated by the addition of 10 μL of Kinase detection reagent (Promega) for 30 min at room temperature. Luminescence signals are determined using appropriate luminescence plate-reader (typical integration time 0.3-1 sec). To calculate percent inhibition, average background signal is subtracted from test well and maximal signal wells. Inhibition, % = (1- (test signal/maximal signal))*100. The percent inhibition at a specified concentration is determined or IC₅₀ values are calculated based on a dose range of inhibitor concentrations using non-linear regression in GraphPad Prism software.

Suebsuwong C., Dai B., Pinkas D.M., Duddupudi A.L., Li L., Bufton J.C., Schlicher L., Gyrd-Hansen M., Hu M., Bullock A.N., Degterev A, & Cuny G.D. (2020). Receptor-Interacting Protein Kinase 2 (RIPK2) and Nucleotide-Binding Oligomerization Domain (NOD) Cell Signaling Inhibitors Based on a 3,5-Diphenyl-2-aminopyridine Scaffold. European journal of medicinal chemistry, 200, 112417.

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PIKfyve Inhibition Kinase Assay

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Recombinant PIKfyve (Carna Biosciences, #11-118) was incubated with inhibitors (SB203580, SB202190, or YM201636) in kinase assay buffer (50 mM HEPES pH 7.5, 50 mM NaCl, 20 mM MgCl₂, 0.5 mM EGTA, 0.04% Triton X-100, 25 µg/mL BSA, 0.05 mM DTT) for 30 min at room temperature (RT). Kinase reactions were initiated by adding 5 µL of a mixture of PI(3)P, phosphatidylserine (PS), and ATP. After 2 h, 5 µL of ADP Glo Reagent (Promega), supplemented with 0.01% Triton X-100, was added and incubated for an additional 40 min. Finally, Kinase Detection Reagent (10 µL) was added, incubated for an additional 30 min, and each sample assayed for luminescence. Final assay concentrations: PIKfyve (1 ng/µL), PI(3)P (50 µM), PS (400 µM), ATP (50 µM), inhibitor (0–2000 nM), and DMSO (0.02%).

Wible D.J., Parikh Z., Cho E.J., Chen M.D., Jeter C.R., Mukhopadhyay S., Dalby K.N., Varadarajan S, & Bratton S.B. (2024). Unexpected inhibition of the lipid kinase PIKfyve reveals an epistatic role for p38 MAPKs in endolysosomal fission and volume control. Cell Death & Disease, 15(1), 80.

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