GTP hydrolysis activities of purified His12-Arf proteins were assayed by quantitating free phosphate molecules released in the hydrolytic reactions, using the Malachite Green-based reagent Biomol Green (Enzo Life Sciences) as described for the activity assays with Rab-family small GTPases (Inoshita and Mima, 2017; Segawa et al., 2019) . Purified His12-Arf1 and His12-Arf6 proteins (2 M final) were mixed with GTP or GTPS (1 mM final) in RB150 containing 5 mM MgCl 2 and 1 mM DTT (100 l each), incubated (30°C, 1 h), and then supplemented with the Malachite Green reagent (100 l each). After incubation with the reagent (30°C, 30 min), the absorbance at 620 nm (A620) of the reactions (200 l each) was measured with a DU720 spectrophotometer (Beckman Coulter). The raw A620 data were corrected by subtracting the A620 values of the control reactions without His12-Arf proteins. Concentrations of phosphate molecules released in the reactions were calculated using the corrected A620 data and the A620 values of phosphate standards (Enzo Life Sciences). Means and standard deviations of the specific GTPase activities (M phosphate/min/M protein) were determined from three independent experiments.
Biomol green
Manufactured by Enzo Life Sciences
Sourced in United States, Germany, Switzerland
Biomol Green is a fluorescent dye used for the detection and quantification of nucleic acids, such as DNA and RNA, in various applications like electrophoresis, flow cytometry, and nucleic acid staining. It exhibits excitation and emission maxima at 500 nm and 520 nm, respectively, and is compatible with common fluorescence detection systems.
Automatically generated - may contain errors
Lab products found in correlation
Adenosine triphosphate (atp), by Merck Group (5 mentions)
Phosphate standard, by Enzo Life Sciences (3 mentions)
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Du720 spectrophotometer, by Beckman Coulter (2 mentions)
Blu002a250uc, by PerkinElmer (2 mentions)
Mgcl2, by Merck Group (2 mentions)
Glucose 1 phosphate, by Merck Group (2 mentions)
Hepes, by Merck Group (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Prescission protease, by Merck Group (1 mentions)
Enspire 2300 multilabel plate reader, by PerkinElmer (1 mentions)
Enzchek kit, by Thermo Fisher Scientific (1 mentions)
Clariostar plate reader, by BMG Labtech (1 mentions)
E coli pyrophosphatase, by New England Biolabs (1 mentions)
384 well microplate, by Greiner (1 mentions)
Pherastar microplate reader, by BMG Labtech (1 mentions)
Spectramax 190 microplate spectrofluorometer, by Molecular Devices (1 mentions)
Imidazole, by Merck Group (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Tris hcl, by Merck Group (1 mentions)
71 protocols using biomol green
1
Quantifying Arf GTPase Hydrolysis
2
Kinase and Phosphatase Assays for Transcriptional Regulation
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Kinase assays were performed with purified proteins (Cdk9/Pch1 complex and GST-Spt5801-990) and [γ-32P] ATP (PerkinElmer, BLU002A250UC), as described previously20 (link). GST-PP1s were expressed in E. coli at 16°C for 16 h and purified with Glutathione Sepharose 4 Fast Flow beads35 (link). To measure protein phosphatase activity, PP1 isoforms (GST-Dis2 or GST-Sds21) purified from E. coli (2 µg) or immunoprecipitated from fission yeast extracts (4 mg total protein) were incubated with 50 µM peptide (Spt5-NP, Spt5-pT1 or H3pS10) at 37°C for 1 h. Colorimetric assays were performed in triplicate using BioMOL Green (Enzo Life Sciences, BML-AK111) in 25 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM MnCl2 and 1 mM DTT, in 96-well plates as described in manufacturer’s protocol. To test Dis2 activity after phosphorylation by Cdk9, Dis2 immunoprecipitated from fission yeast extract (8 mg total protein) was incubated with Cdk9 (activated by Csk1) or mock-treated (no kinase added) in kinase assay buffer (25 mM HEPES, pH 7.5, 10 mM MgCl2, 1 mM ATP, 1 mM DTT) for 1 h at 25°C, washed three times with phosphatase assay buffer and tested for activity as described above.
3
Kinase and Phosphatase Assays for Transcriptional Regulation
4
ATPase Activity Assay for TOP2B
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ATPase activity assays were performed using BioMOL green (Enzo)(Rule et al. 2016 ). The assays were performed in a reaction mixture containing 50 mM Tris HCl pH 7.5, 50 mM KCl, 10 mM MgCl2, and 0.1 mM ATP (unless otherwise stated). TOP2B ATPase domain proteins were then added to start the reaction and incubated at 37°C for 30 min. The concentrations of TOP2B were varied as detailed in figure legends. For DNA stimulation experiments, 4 μg pBR322 DNA was added prior to TOP2B. 50 μL of the reaction was then transferred to a microtitre plate containing 100 μL BioMOL green to terminate the experiment. After 20 min the absorbance was measured at 655 nm. Reactions containing no enzyme were performed to generate a background reading of inorganic phosphate and were subtracted from the experimental results. The inorganic phosphate released was then calculated based on the absorbance standard curve established by phosphate standards. The kinetic parameters Km and Vmax were calculated from the Lineweaver-Burk plots using GraFit. All experiments were repeated at least three times.
5
MinD-MinE ATPase Activity Assay
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ATP hydrolysis was measured by monitoring the amount of inorganic phosphate released after 10 min in reactions containing Tris buffer (20 mM; pH 7.5), KCl (50 mM), MgCl2 (10 mM), ATP (4 mM), MinD (8 μM), MinD(R3E) (8 μM), MinE (16 μM), and SUVs (1 mg ml−1), where indicated. Phosphate released in reactions was detected using Biomol green (Enzo Life Sciences) and compared with a phosphate standard curve.
6
Quantifying VCP ATPase Activity
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For ATPase activity of VCP 500 ng of recombinant active GST-VCP (SignalChem, #VCP-195H) was incubated in 50 µL of reaction buffer (10 mM HEPES-KOH pH 7.7, 2.5 mM MgCl2, 50 mM KCl, and 1 mM DTT) together with DMSO, 20 µM SMER28 or DBeQ, NMS873, CB-5083. The reaction was started by the addition of 0,1 mM ATP and carried out for 1 h at room temperature. The reaction was stopped by the addition of 100 µL BIOMOL green (Enzo LifeSciences, #BML-AK111-0250), and after 30 min absorbance at 650 nm was determined and the amount of released phosphate was interpolated from a standard curve. For ATPase activity of WT VCP, ATPase mutants and the VCP-R155H mutant, GST-tagged VCP proteins were purified from E. coli (GST-tag removed by PreScission protease; Merck, #GE27-0843-01), and used in ATPase assay buffer (20 mM HEPES-KOH pH 7.7, 20 mM MgCl2, 50 mM KCl, and 1 mM DTT). Reaction was started by the addition of 2 mM ATP and carried out at 37 °C for denoted time points, followed by the addition of 100 µL BIOMOL green and absorbance reading. 200–500 ng of purified protein was used per reaction.
7
Quantifying ATPase Activity of ClpC1
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ATPase activity of ClpC1 was determined by monitoring the released phosphate level using BIOMOL® Green (Enzo Life Science, Farmingdale, NY, USA). The development of green color intensity was monitored via absorbance at 620 nm. The amount of phosphate released was converted using a phosphate standard curve. A graph of phosphate released versus ATP concentration was plotted with the OriginPro 8 program. The curve was fitted using Hill equations as follows: y = START + (END-START) × xn/(kn + xn) (Ito, Akiyama, 2005). Statistical analysis was performed with OriginPro8 and Excel software. Data were expressed as mean ± standard error (SD) or frequency (%). Ecumicin and rufomycin (provided by the Nutrition and Pharmaceutical Center, Yongin, Republic of Korea) were used as a positive control in this screening study [14 (link),35 (link),36 (link)].
8
ATP Hydrolysis Rates of ClpX
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ATP hydrolysis rates for ClpX (0.5 μM) in HEPES buffer (50 mM, pH 7.5), with KCl (150 mM), MgCl2 (20 mM), and 5 mM ATP in the absence and presence of Gfp-IDRFtsZ, Gfp-IDRFtsZ mut-1 and Gfp-IDRFtsZ mut-2 (all 8 μM) were determined by measuring the amount of free phosphate released with time (0, 5, 10 and 15 minutes) using Biomol Green (Enzo Life Sciences). Phosphate was quantified by comparison to a phosphate standard curve.
9
Standard ATPase Activity Assay
10
Enzymatic Activity Monitoring Protocols
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The hydrolysis rates and inhibition constants
for adenosine deaminase and MTA-SAH deaminase activities were monitored
spectrophotometrically in a direct assay. Inhibition of isoaspartyl
dipeptidase was measured in a spectrophotometric assay coupling the
formation of aspartate to the oxidation of NADH. The substrate hydrolysis
rates and inhibition constants for phosphoserine phosphatase were
measured spectrophotometrically using the Enzchek kit (Invitrogen).
The hydrolysis rates and inhibition constants for flavin mononucleotide
phosphatase were monitored using either the Enzchek kit or BIOMOL
Green (Enzo Life Sciences). The hydrolysis of AmpC β-lactamase
substrates and enzyme inhibition were monitored spectrophotometrically
(methods in theSI ).
for adenosine deaminase and MTA-SAH deaminase activities were monitored
spectrophotometrically in a direct assay. Inhibition of isoaspartyl
dipeptidase was measured in a spectrophotometric assay coupling the
formation of aspartate to the oxidation of NADH. The substrate hydrolysis
rates and inhibition constants for phosphoserine phosphatase were
measured spectrophotometrically using the Enzchek kit (Invitrogen).
The hydrolysis rates and inhibition constants for flavin mononucleotide
phosphatase were monitored using either the Enzchek kit or BIOMOL
Green (Enzo Life Sciences). The hydrolysis of AmpC β-lactamase
substrates and enzyme inhibition were monitored spectrophotometrically
(methods in the
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