Recombinant p97/VCP was diluted in assay buffer (50 mmol/L Tris‐HCl [pH 8.0], 20 mmol/L MgCl, 1 mmol/L EDTA, 1 mmol/L DTT) to a final concentration of 0.5 μmol/L. Then, 72 μL of the mixture was dispensed into a 96‐well plate and 4 μL of compound stocks of various concentrations of OSSL_325096 or DMSO was added to each well. The plate was incubated for 10 minutes at room temperature. Then, 10 μL of 0.5 mmol/L ATP solution was added to each well and incubated for 30 minutes at room temperature. ATPase activity was quantified using a QuantiChrom ATPase/GTPase Assay Kit (BioAssay Systems, Hayward, CA, USA).
Quantichrom atpase gtpase assay kit
Manufactured by BioAssay Systems
Sourced in United States
The QuantiChrom ATPase/GTPase Assay Kit provides a sensitive and convenient method for the quantitative determination of ATPase and GTPase activities in biological samples. The kit utilizes a chromogenic reagent that directly measures the phosphate generated from the enzymatic hydrolysis of ATP or GTP. The assay is simple, fast, and can be readily adapted for high-throughput applications.
Automatically generated - may contain errors
Lab products found in correlation
Versamax elisa microplate reader, by Molecular Devices (1 mentions)
Flexstation 3, by Molecular Devices (1 mentions)
Biotek synergy neo plate reader, by Agilent Technologies (1 mentions)
Prism 5.0 program, by GraphPad (1 mentions)
Cutsmart buffer, by New England Biolabs (1 mentions)
Synergy 4, by Agilent Technologies (1 mentions)
16 protocols using quantichrom atpase gtpase assay kit
1
Quantification of p97/VCP ATPase Activity
2
Quantifying GTPase Activity of TOC34
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To investigate the GTPase activity of TOC34, we performed GTPase assay using a Quantichrom™ATPase/GTPase assay kit (BioAssay Systems, USA). The GTP hydrolysis activity of the TOC34 protein purified from E. coli was determined according to manufacturer's instructions. Briefly, the assays were initiated by adding 10 μL of TOC34 (2.5 μg) into 30 μL reaction samples containing 20 mM Tris (pH 7.0), 40 mM NaCl, 4 mM MgAc2, 0.5 mM EDTA, 4 mM GTP, and 5 mM CaCl2 or 2 mM EGTA. Depending on reaction conditions, either 2.1 μg of CBL10 or 1.8 μg of CBL4 was also included. After incubation at 27°C for 30 min, free phosphates generated were quantified according to the manufacturer's protocols using a VersaMax ELISA microplate reader (Molecular Devices, USA).
3
Quantifying ZIKA NS3 ATPase Activity
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The ATPase activity was detected with QuantiChrom™ATPase/GTPase Assay Kit (BioAssay Systems). The free phosphate released by ATP hydrolysis was quantified with the reagent in the kit to indicate the ATPase activity. Phosphate standards were prepared according to the manual. The reaction was performed in a final volume of 40 μl in 96-well plates. 30 nM wild type or mutated ZIKA NS3 protein were incubated with various concentrations of ATP in buffer (20 mM Tris, 40 mM NaCl, 4 mM Mg(AcO)2, 0.5 mM EDTA, pH 7.5) at RT. Equal amount of dialysis buffer without protein was used as control. The ATP hydrolysis reaction was terminated at 30 min by adding 200 μl reagent. After incubation for another 30 min at room temperature, the absorbance was measured at 620 nm on a plate reader (Flexstation3, Molecular Devices). The reaction velocity and ATP concentration were fitted to the Michaelis–Menten equation (V = Vmax[S]/Km + [S]). For the ATP hydrolysis assay with DNA or RNA substrates, 30 nM or 3 μM ssDNA or ssRNA was used in the experiment as indicated. Sequences of DNA and RNA are shown below:
4
Hsp90α ATPase Activity Assay
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In brief, ATPase activity assay was performed by mixing Hsp90α solution (5 μL, 6 μM in assay buffer), 20 μL assay buffer, ATP solutions (10 μL, 4 mM in assay buffer), and 5 μL of tested compounds (14 and BIIB021) to yield final concentrations of 50 μM for 30 min at the room temperature using the QuantiChrom ATPase/GTPase Assay Kit (BioAssay Systems, Hayward, CA, USA). The reaction was terminated by adding 200 μL reagent buffer. Followed by incubation with reagent buffer for 30 min at the room temperature, the absorbance was measured at 620 nm using a Biotek-Synergy Neo-Plate Reader (BioTek Instruments, Winooski, VT, USA).
5
ATPase Activity Assay of hPMCA1 Proteins
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The hPMCA1-NPTN and hPMCA1 alone proteins used for ATPase activity assay were purified as described above. The ATPase activity was measured using QuantiChrom ATPase/GTPase assay kit (BioAssay Systems). The protein concentrations for the assays ranged from 0.05 to 0.2 mg/ml. All reactions were performed using the reaction buffer from the assay kit with final concentration of 1.83 mM CaCl2, 5 mM MgCl2, 1.75 mM EDTA, 0.05% digitonin, 1 mM DTT, and indicated ATP. Reactions were carried out at 37 °C for 10 min and stopped by addition of the reagent from assay kit. The mixture was incubated for 30 min at room temperature before the activity was measured by monitoring the increase of absorbance at 620 nm. Nonlinear regression to the Michaelis-Menten equation and data analysis was performed using OriginPro 8.
6
ZIKV NS3 Helicase ATPase Activity Assay
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The ATP activity assay was carried out using the QuantiChrom™ ATPase/GTPase Assay Kit (BioAssay Systems, Hayward, CA, USA) by following product instructions. The ZIKV NS3 helicase was preincubated at a concentration of 40 nM in 20 μl assay buffer (40 mM Tris, 80 mM NaCl, 8 mM Mg(AcO)2, 1 mM EDTA, pH 7.5 or 40 mM Tris, 80 mM NaCl, 1 mM EDTA, pH 7.5) in a 96-well plate. The reaction was carried out with ATP or GTP at a concentration of 0.5 mM for various time at room temperature and then terminated by adding 200 μl of reagent buffer. Followed by incubation with reagent buffer at the room temperature, the absorbance was measured at 630 nm.
7
Intrinsic GTPase Activity Assay
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Small GTPase assay was performed according to our previous report (Xue et al., 2020 (link)). Intrinsic GTP hydrolysis of the bacterially expressed BBS3, BBS3A73L, and BBS3T31R was measured by optical assay for the release of inorganic phosphate with reagents from the QuantiChrom ATPase/GTPase assay kit (Bioassay Systems) (Pan et al., 2006 (link)).
8
NS3 Helicase ATPase Activity Assay
9
Quantichrom ATPase/GTPase Assay for RvhD4
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RvhD4 ATPase activity was monitored using a Quantichrom ATPase/GTPase assay kit (Bioassay Systems), according to the manufacturer’s instructions and as described previously [113 (link)]. Briefly, 200–12.5 ng/well of purified recombinant RvhD4 protein was incubated in the presence of 1 mM ATP for 30 min at 37°C. Generated free phosphate was quantified by measuring absorbance at OD 620 nm. All of the samples were measured in triplicate wells, and data are given as averages ± S.D. of three independent experiments.
10
Quantifying ATPase/GTPase Activity
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The ATPase/GTPase activity was measured using QuantiChrom ATPase/GTPase assay kit (BioAssay Systems). All reactions were performed in the reaction buffer from the assay kit with various concentrations of ATP/GTP indicated and 0.2 μM protein at 37 °C for 30 min. Then follow the steps of the kit manual and the light absorbance of sample was measured at 620 nm. Nonlinear regression to the Michaelis-Menten equation and statistical analysis were performed using GraphPad Prism 8.
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