Enzyme activity of MARK4 was evaluated with the help of standard Malachite Green (Biomol Green reagent, Enzo Life Sciences) microtitre-plate assay as described earlier41 (link). Estimated amount of MARK4 (200 ng) and 20 µM ATP as a substrate were incubated for 10–15 min at 25 °C with increasing concentrations (0–20 µM) of synthesized acridone derivatives, in 20 mM Tris buffer, pH 8.0. Reaction was terminated by the adding 100 µl of Biomol Green reagent and absorbance of final reaction products was recorded at 620 nm in a multiplate ELSIA reader (BioRad). In order to quantify the hydrolyzed phosphate, inorganic phosphate standard curve were prepared from the phosphate standard solutions as supplied by the manufacturer (Biomol Green reagent, Enzo Life Sciences). All data points represent triplicate measurements from at least three independent experiments.
Biomol green reagent
Manufactured by Enzo Life Sciences
Sourced in United States
BIOMOL Green reagent is a fluorescent dye used for the detection and quantification of proteins in various laboratory applications. It binds to proteins and emits a green fluorescent signal when excited by light of the appropriate wavelength.
Automatically generated - may contain errors
Lab products found in correlation
Phosphate standard, by Enzo Life Sciences (3 mentions)
Infinite m200 plate reader, by Tecan (3 mentions)
Spectramax m5, by Molecular Devices (2 mentions)
200 plate reader, by Tecan (2 mentions)
Adenosine triphosphate (atp), by Merck Group (2 mentions)
Synergy neo2, by Agilent Technologies (2 mentions)
Synergy neo2 plate reader, by Agilent Technologies (2 mentions)
Ni nta resin, by Thermo Fisher Scientific (2 mentions)
Prism 7, by GraphPad (2 mentions)
Multiscan ex, by Thermo Fisher Scientific (1 mentions)
Infinite m1000 pro, by Tecan (1 mentions)
Wallac victor2 plate reader, by PerkinElmer (1 mentions)
Synergy neo microplate reader, by Agilent Technologies (1 mentions)
Adenosine triphosphate (atp), by Roche (1 mentions)
Infinite m1000 pro multimode plate reader, by Tecan (1 mentions)
Clariostar plate reader, by BMG Labtech (1 mentions)
αβ methylene adp, by Merck Group (1 mentions)
Enspire, by PerkinElmer (1 mentions)
Prism v10, by GraphPad (1 mentions)
Adenosine triphosphate (atp), by Thermo Fisher Scientific (1 mentions)
62 protocols using biomol green reagent
1
Malachite Green Assay for MARK4 Activity
2
ATPase Activity Assay of p97 Protein
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Detection of ATPase activity using Biomol Green reagent (Enzo Life Sciences, Farmingdale, NY, USA) was performed as previously described with slight modifications [45 (link)]. To compare the ATPase activity of wild type and mutant p97 proteins, each purified protein was diluted to a final monomer concentration of 25 nM in 50 μL ATPase assay buffer (50 mM Tris (pH 7.4), 20 mM MgCl2, 1 mM EDTA, 0.5 mM TCEP, 0.01% Triton X-100 and 80 nM BSA). After adding 200 μM ATP, the reaction was carried out at room temperature for optimal reaction times. 50 μL Biomol Green reagent (Enzo Life Sciences, Farmingdale, NY, USA) was added to stop the reaction and the absorbance at 635 nm was measured using a BioTek Synergy Neo 2 plate reader (BioTek, Winooski, VT, USA). The eleven-dose titrations with p47 cofactor were performed by adding the varying amount of p47 protein in ATPase buffer with p97. The results were calculated from five replicates using GraphPad Prism 7.0.
3
Trypanothione Synthetase Enzyme Assay
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TryS activity was determined by the end-point assay20 (link), which is described below. For all TryS, ATP was used at 150 µM and SP fixed at 2 mM, whereas GSH was adjusted to 0.57, 0.25 and 0.05 mM for TcTryS, LiTryS and TbTryS, respectively, to avoid substrate inhibition or to approach physiological concentrations. A master mix (MM) solution containing all the substrates at 1.25-fold their end concentration in assay was prepared in the screening reaction buffer (5 mM DTT, 10 mM MgSO4, 0.5 mM EDTA, 100 mM HEPES pH 7.4). Ninety-six wells microtitre plates were loaded with 5 µL/well of the test compounds, DMSO (reaction control) or TryS-specific inhibitor (inhibition control), and 40 µL of MM. The reactions were started by adding 5 µL of TryS and stopped after 15 min with 200 µL BIOMOL GREENTM reagent (Enzo Life Sciences, Farmingdale, NY). The plates were incubated 20 min at room temperature and then A650 nm was measured with a MultiScan EX plate reader (Thermo Fisher Scientific, Waltham, MA). Blanks were prepared for each condition by adding 5 µL of the screening reaction buffer instead of enzyme. The interference of the compounds with the colorimetric reaction was evaluated in a sample containing different concentrations of the compound and 20 µM K2HPO4 dissolved in MM. The activity values were corrected accordingly. All determinations were performed at least by quadruplicates.
4
Prp43 Helicase Enzymatic Assay
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Reaction mixtures (50 μl) contained 10 nM Prp43 (wild-type or mutant), 0.2 mM polyA, 100 mM KCl, 25 mM HEPES–KOH pH 8, 2.5 mM Mg(CH3COO)2 and 0.2 mM DTT. Enzymatic assays were carried in 96-well microplate and free phosphate assays were performed with BIOMOL GreenTM reagent (Enzo Life Science). At each time, reaction was quenched by the addition of 0.5 M EDTA pH 8.8. At the end of the experiment, 200 μl BIOMOL GreenTM reagent were added in each well, the absorbance at 620 nm was monitored after an incubation of 25 min at 25°C. The quantity of free phosphate was calculated by interpolations of the values on a phosphate standard curve. All measurements were performed in triplicates. Kinetic parameters were determined by non-linear fit.
5
ATPase activity of CtCCT variants
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ATPase activities were measured at 30, 35 and 40°C in TKM buffer (50 mM Tris-HCl, pH 7.5, 100 mM KCl, and 25 mM MgCl2) containing 1 mM ATP and 20 μg/ml CtCCT variants. Phosphate ion production was measured using the malachite green assay with BIOMOL GREENTM reagent (Enzo life Sciences).
6
Phosphatase Activity Assay Protocol
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Recombinant PTP domains were made up to 20 μl volumes in a 96-well microplate format in reaction buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 5% glycerol, 5 mM DTT). Thirty microliters of 100 μM pSer, pThr, pTyr peptides (DADE-pY-LIPQQG and END-pY-INASL) or imidodiphosphate was added to protein wells to initiate reactions. Reactions were allowed to proceed for 15 min before termination by addition of 100 μl BIOMOL Green reagent (Enzo). Liberated phosphate was measured by absorbance at 360 nm in a Spectramax M5 plate reader (Molecular Devices), followed by interpolation to a standard curve of known phosphate concentration. Serial dilutions of phosphate were performed in reaction buffer using 800 μM phosphate standard (Enzo).
7
Colorimetric ATPase Activity Assay
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A malachite green/molybdate‐based colorimetric assay (Biomol Green reagent, Enzo Life Sciences) was used to assess ATPase activity as described previously by us and others (D'Arcy et al., 2019 (link); Rule et al., 2016 (link)). Briefly, reactions containing 20 μM enzyme were assembled in 20 mM HEPES pH 8.5, 13 mM NaCl, 1% glycerol, 5 mM ATP, and 5 mM MgCl2 in a total volume of 30 μl. Reactions were initiated by the addition of ATP and MgCl2, and at the indicated time points (0, 15, 30, 45, and 60 min), 5 μl aliquots were removed and quenched by diluting into 245 μl 1 × HNG buffer followed by flash freezing in liquid nitrogen. To determine the amount of orthophosphate released, Biomol Green reagent was added to each well and absorbance was measured at 620 nm using a Tecan Infinite M1000 Pro multimode plate reader. The mean ± SEM was derived from the cumulative data generated from three separate experiments.
8
PTEN Phosphatase Activity Assay
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Water-soluble diC8-PI(3,4,5)P3 (Echelon) was used to measure PTEN phosphatase activity against PI(3,4,5)P3. In all, 250 mM recombinant PTEN was reacted with 5 nmol diC8-PI(3,4,5)P3 at 21 °C for 2 h with gentle shaking in 50 μL of PTEN storage buffer. After the reaction, 150 μL of BIOMOL® green reagent (ENZO) was added, and the mixture was shaken gently for 1 h. In order to quantify the phosphate released from the substrate by the dephosphorylation reaction, the absorbance at 620 nm was measured.
9
Inhibition of Human p97 ATPase Activity
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The detailed method was described previously.28 (link) Inhibition of human p97 (25 nM monomer) was carried out in assay buffer (50 mM Tris pH 7.4, 20 mM MgCl2, 1 mM EDTA, 0.5 mM TCEP) containing 0.01% Triton X-100 and 200 μM ATP. Eight-dose titration was used to determine IC50 of each compound in blocking ATPase activity, which was determined through the addition of Biomol Green Reagent (Enzo Life Sciences, Farmingdale, NY, USA).
10
Phosphatase Kinetic Assay Protocol
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Phosphatase assays were carried out as previously described (Touzé et al., 2008a (link)). Assays were performed with a total volume of 50 µl in buffer containing 20 mM Tris/HCl pH 7.5, 150 mM NaCl, 10 mM β-mercaptoethanol, 0.02% DDM, and 10 mM MgCl2. The substrate farnesyl pyrophosphate (Millipore Sigma) was added at a range of concentrations from 5 to 80 µM. The enzyme concentrations were 42 nM PgpB, 42 nM BacA, and 66 nM PBP1B. After incubation for 15 min at ambient temperature 140 µL Biomol Green reagent (Enzo Life Sciences, BML-AK111-0250) was added and samples were incubated for 30 min at ambient temperature to allow for colour development. Absorbance at 620 nm was measured in a SpectraMax M2 spectrophotometer. Inorganic phosphate standard provided with the Biomol Green reagent was used to obtain a standard curve that was used to convert enzyme assay absorbance into phosphate concentration. The enzyme kinetics curves were analysed using classical Michaelis–Menten equation using the Excel add-on, Solver.
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