The sequences of FgRab1 cDNA, FgGyp1 cDNA, TBC domain and R357K point mutant of FgGyp1 were cloned into the MBP (Maltose-binding protein) vector pMAL-c2X, using their respective primers listed in Supplementary Table 2 , and expressed. The protein products were isolated and purified for GAP activity assay using a GTPase assay kit (Sigma-Aldrich, Catalog Number MAK113) according to the manufacturer’s protocols. GTP hydrolysis was assayed based on previous reports (Xiong et al., 2012 (link); Zheng et al., 2020 (link)). Briefly, the recombinant proteins MBP–FgGyp1, MBP–FgGyp1TBC (TBC), MBP–FgGyp1R357K (R357), and MBP–FgRab1 were expressed in BL21 Escherichia coli strain and isolated by Amylose resin (Sangon Biotech, NO.C500096) as per the instructions of the manufacturer. A colorimetry-based kit was used (Sigma-Aldrich, Catalog Number MAK113) to assay for the GTPase activity of Gyp1, and FgRab1 was incubated with FgGyp1, FgGyp1TBC (TBC), FgGyp1R357K (R357K), and MBP control, respectively, following the instructions of the manufacturer. The experiment was repeated three times.
Mak113
Manufactured by Merck Group
MAK113 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use. The core function of MAK113 is to perform sample preparation and processing tasks, however, a detailed description of its intended use cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Gtpase assay kit, by Merck Group (2 mentions)
Amylose resin, by Sangon (2 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Celltiter glo 2, by Promega (1 mentions)
Lipofectamine 2000 reagent protocol, by Thermo Fisher Scientific (1 mentions)
Spark 10m, by Tecan (1 mentions)
Arvo x3, by PerkinElmer (1 mentions)
Biotek synergy h4 hybrid multi mode microplate reader, by Agilent Technologies (1 mentions)
Spectramax m5, by Molecular Devices (1 mentions)
10 protocols using mak113
1
GTPase Activity Assay for FgRab1 and FgGyp1
2
Biochemical Analysis of Fungal Rab1 GTPase Regulation
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The sequences of FgRab1 cDNA, FgGyp1 cDNA, TBC domain and R357K point mutant of FgGyp1 were cloned into the MBP (Maltose-binding protein) vector pMAL-c2X, using their respective primers listed in Supplementary Table 2, and expressed. The protein products were isolated and purified for GAP activity assay using a GTPase assay kit (Sigma-Aldrich, Catalog Number MAK113) according to the manufacturer's protocols. GTP hydrolysis was assayed based on previous reports (Xiong et al., 2012; (link)Zheng et al., 2020) (link). Briefly, the recombinant proteins MBP-FgGyp1, MBP-FgGyp1 TBC (TBC), MBP-FgGyp1 R357K (R357), and MBP-FgRab1 were expressed in BL21 Escherichia coli strain and isolated by Amylose resin (Sangon Biotech, NO.C500096) as per the instructions of the manufacturer. A colorimetry-based kit was used (Sigma-Aldrich, Catalog Number MAK113) to assay for the GTPase activity of Gyp1, and FgRab1 was incubated with FgGyp1, FgGyp1 TBC (TBC), FgGyp1 R357K (R357K), and MBP control, respectively, following the instructions of the manufacturer. The experiment was repeated three times.
3
Purification and Characterization of OsRPT2a Variants
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The coding sequences for OsRPT2a and OsRPT2aK240A were each fused to the glutathione S-transferase (GST) tag in the vector pGEX-KG (GenBank accession LT986714.1) at the EcoRI and HindIII sites, to produce the constructs for GST fusions. The constructs were transformed into E. coli strain BL21 competent cells and the target proteins were induced by isopropyl-β-d -thiogalactoside at a concentration of 0.3 mM. The fusion targets were purified through affinity chromatography using Glutathione Sepharose 4B (GE Healthcare, NJ, USA). Details for ATPase activity assays are described in the technical bulletin for MAK113 of Sigma-Aldrich Co. LLC. (St Louis, MO, USA).
4
Quantification of Cellular ATP Levels
5
Characterization of MYO6 Missense Variants
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HeLa cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM)/high-glucose medium with 10% fetal bovine serum and supplemented with penicillin–streptomycin–glutamine. Cells were maintained in a humidified incubator at 37 °C and 5% CO2. Twenty-four hours before transfection, cells were trypsinized and seeded into a 6-well plate at a density of 3 × 105 cells per well to reach 60–80% confluency. The lipofectamine 2000 reagent protocol (Invitrogen Life Technologies) was used for transfecting cells with 4 µg of DNA plasmid carrying wild type and missense variant.
Total protein was extracted and loaded in SDS-PAGE gels as described previously, and the membrane was blotted using MYO6 MYO6 (M0691, Sigma-Aldrich, St. Louis, MO, USA) and (PA5-35054, Thermo Fisher Scientific, Waltham, MA, USA), and monoclonal anti-α-tubulin (T5168, Sigma-Aldrich) antibodies.
ATPase assays were performed by using a malachite green-based colorimetric assay (MAK113, Sigma-Aldrich) according to the manufacturer’s protocol.
Total protein was extracted and loaded in SDS-PAGE gels as described previously, and the membrane was blotted using MYO6 MYO6 (M0691, Sigma-Aldrich, St. Louis, MO, USA) and (PA5-35054, Thermo Fisher Scientific, Waltham, MA, USA), and monoclonal anti-α-tubulin (T5168, Sigma-Aldrich) antibodies.
ATPase assays were performed by using a malachite green-based colorimetric assay (MAK113, Sigma-Aldrich) according to the manufacturer’s protocol.
6
ATPase Activity Assay for Protein Subunits
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ATPase activities for the wild-type A- and B-subunit monomers and the designed B-subunit monomer were measured with an ATPase/GTPase activity assay kit (MAK113, Sigma-Aldrich). Proteins (250 nM) were incubated with 1 mM ATP in 40 μl assay buffer (40 mM Tris, 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA, pH 7.5) in 96-well plates (Greiner, 655801) for 1 h at room temperature. Solutions without protein samples were incubated as the reference at the same condition. After the incubation, 200 μl malachite green reagents were added to each well and the solutions were incubated for 30 min at room temperature. For the incubated solutions, absorbances at 350–850 nm were measured with a Spark 10M (TECAN) microplate reader. Product (phosphate) concentrations were estimated from the relative absorbance (620 nm) for the reference by comparing with the absorbances for standard buffers at several phosphate concentrations. ATPase activity for the A subunit was calculated from the estimated product concentrations.
7
Measuring Top2 ATPase Activity
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S. pombe Top2 proteins were immunoprecipitated as described above and subjected to a malachite green assay using an ATPase/GTPase activity assay kit (Sigma, MAK113). Kinetoplast DNA (270 ng) was incubated with the Top2 IP fraction (0.1 μg) in assay buffer containing 40 mm Tris, 80 mm NaCl, 8 mm MgAc2, and 1 mm EDTA (pH 7.5). Assays were performed in the presence or absence of 5 μm ICRF-193 at 30 °C for 30 min in triplicate. The resulting colorimetric product was measured at 600 nm with a spectrophotometric plate reader (ARVO X3, PerkinElmer Inc.). The immunoprecipitated fraction from a strain expressing an empty vector was also assayed and used as a background control. Background readings could then be subtracted from sample readings.
8
ATP Hydrolysis Kinetics of PAN Proteasome
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The rate of ATP hydrolysis by PAN was measured offline in the same conditions as the SANS experiments. For this purpose, the amount of inorganic phosphate was recorded as a function of time. Reaction mixtures were prepared with hydrogenated proteins (70 μM GFPssrA, 20 μM PAN, and 20 μM 20S) in an H2O buffer containing 20 mM Tris-HCl (pH 7.5), 100 mM NaCl, 100 mM ATP, and 200 mM MgCl2. The following conditions were measured in triplicate: isolated PAN, PAN and GFP, PAN, 20S and GFP, isolated GFP, isolated 20S, and buffer. The samples were incubated at 55°C and monitored over 1 h with a 20 μL reaction mixture measured at each time point. The aliquots were incubated on ice to stop the reaction and diluted 150 times in the buffer. 4 μL were then supplemented with 36 μL of assay buffer and 200 μL of malachite green reagent (MAK113; Sigma-Aldrich). After 30 min of incubation at room temperature, absorbance at 630 nm was analyzed by using a plate reader spectrophotometer (BioTek Synergy H4 Hybrid Multi-Mode Microplate Reader; BioTek Instruments). A standard phosphate curve was used to estimate the amount of phosphate released during the hydrolysis of ATP. Phosphate concentrations were fitted with a biexponential process (Eq. 1 ) in which intensities were replaced by concentrations.
9
ATPase Activity Assay in A. citrulli
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ATPase activity assay was performed using an ATPase/GTPase activity assay kit according to the manufacturer’s instructions (MAK113, Sigma). A. citrulli was prepared as described above; the cells were harvested by centrifugation at 13,000 rpm for 10 min at 4°C, and the pellets were suspended in assay buffer [40 mM Tris buffer (pH 7.5), 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA]. Ten µL of 4 mM ATP was added to 20 µL of cell suspension, and the cells were incubated for 30 min at 25°C. Subsequently, 200 µL of reagent was added to each well, and the samples were incubated for another 30 min at room temperature. Their absorbance was measured at 620 nm using a microplate reader. The experiment was conducted twice with five replicates each.
10
Quantifying ATPase Activity in Thermophilic Mfd
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ATPase activity assays were performed in a 96-well microplate format using a commercial kit (MAK113, Sigma-Aldrich, Inc.). Reaction mixtures contained (40 μl): 5 μM T. thermophilus Mfd, 0.4–2 mM ATP, 50 mM Tris–HCl (pH 7.9), 0.1 M KCl, 10 mM MgCl2, 1 mM DTT and 5% glycerol. Reaction mixtures were incubated 60 min at 37°C. 200 μl reagent (MAK113A, Sigma-Aldrich, Inc.) was added to terminate the enzyme reaction and generate the colorimetric product. The absorbance at 620 nm was measured using a SpectraMax M5 microplate reader (Molecular Devices, Inc.). Phosphate standard solution was used to calculate the extinction coefficient of the colorimetric product. Less than 2% ATP was consumed to make sure initial velocity was measured.
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