The enzymatic activity for ATP hydrolysis was measured by quantifying the phosphate released from ATP using ATPase/GTPase Activity Kit (Sigma-Aldrich). For the assays, FPLC fractions representing the specific multimeric states of DctDD57Q and the d-IIAGlc/DctDD57Q complex were diluted to 10 µmole/l with assay buffer (20 mM HEPES [pH 7.0], 150 mM NaCl, 5% glycerol, and 5 mM MgCl2) and mixed with 5 mM ATP. After the reaction mixtures (80 µL) were incubated at 37°C for 10 min, the mixtures were added with malachite green reagent (20 µL) and subjected to spectrophotometry at 620 nm (11 (link)). As a control, an ATPase-deficient DctD, DctDH216R, was purified from E. coli JM109 carrying pQE30-dctDH216R after treated with alkaline-phosphatase (AP) as described (40 (link)). Then, dephosphorylated form of DctDD216R (d-DctDD216R) and d-IIAGlc complex was dissolved in a buffer (50 mM Tris-HCl [pH 8.0], 20 mM KCl, 50 mM MgCl2, and 100 mM NaCl) and applied to the AKTA-FPLC system equipped with a Superdex 200 Increase 10/300 Gl column. Fractionated dimeric d-DctDH216R and d-IIAGlc/d-DctDH216R complexes were used for ATPase assay.
Atpase gtpase activity kit
Manufactured by Merck Group
The ATPase/GTPase Activity Kit is a laboratory tool designed to measure the activity of ATPase and GTPase enzymes. It provides a reliable and quantitative method to determine the enzymatic activity of these important molecular motors, which play crucial roles in various cellular processes.
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3 protocols using atpase gtpase activity kit
1
ATP Hydrolysis Activity Assay
2
ATPase Activity Assay of BcsRQ Complexes
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ATPase activity assays were conducted using the ATPase/GTPase activity kit (MAK113, Sigma-Aldrich) according to the manufacturer’s guidelines. Following IMAC and SEC purification, wild-type and mutant BcsRQ complexes were diluted to final concentrations of 5, 2.5, 1.25, and 0.625 μM in gel filtration buffer [20 mM Hepes (pH 8.0), 120 mM NaCl, and 2 mM DTT]. Ten microliters of each sample was mixed with 20 μl of the provided assay buffer [40 mM tris-HCl (pH 7.5), 80 mM NaCl, 8 mM MgAc2, and 1 mM EDTA] and 10 μl of 4 mM freshly prepared ATP in a microplate. The reactions were incubated for 25 min at room temperature before stopping them by the addition of the malachite green–containing blocking reagent. The samples were left to develop for additional 25 min at room temperature to allow the malachite green to form a stable dark green product with the free phosphate liberated by the ATPase reactions. The colorimetric products, proportional to enzyme activity, were measured in a microplate reader at 620 nm. A previously characterized active ATPase [FleQT149E (40 (link))] was used as a positive control, and negative control wells contained gel filtration buffer in lieu of protein sample.
3
NLRP3 Inflammasome Activation Assay
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