The ATPase activity of βγ-CAT was measured by an ATPase activity assay kit (Sigma-Aldrich, Cat MAK113-1 KT) according to the manufacturer’s protocol. Briefly, various concentrations βγ-CAT was incubated with 4 mM ATP in assay buffer (40 mM Tris, 80 mM NaCl, 8 mM MgAc2, and 1 mM EDTA, pH 7.5) for 30 min at 26°C. Cell lysates from HepG2 cells were used as a positive control. Absorbance was read at 620 nm, and the free phosphate concentration was calculated by a standard curve (0–50 μM free phosphate).
Atpase activity assay kit
Manufactured by Merck Group
The ATPase Activity Assay Kit is a laboratory product developed by Merck Group. The kit is designed to measure the enzymatic activity of ATPases, a class of enzymes that catalyze the hydrolysis of ATP to ADP and inorganic phosphate. The kit provides the necessary reagents and protocols to quantify ATPase activity in a rapid, sensitive, and reproducible manner.
Automatically generated - may contain errors
12 protocols using atpase activity assay kit
1
ATPase Activity Assay of βγ-CAT
2
ATPase Activity Assay Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ATPase activity was measured following the instructions of ATPase Activity Assay Kit (MAK113, Sigma) as previously reported (Kang et al. 2022b (link)). The reaction was incubated at room temperature for 30 min, and terminated by addition of malachite green reagent. The colorimetric product was measured at 620 nm. Phosphate standards were performed in parallel.
3
Chloroplast ATPase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Intact chloroplasts were isolated from 4-week-old plants according to Mao et al.23 (link). The ATPase activity was determined by measuring by the amount of inorganic phosphate (Pi) in the reaction using an ATPase Activity Assay Kit (Sigma–Aldrich). Isolated chloroplast suspension (10 ul) was incubated for 30 min at room temperature in 30 ul assay buffer containing 40 mM Tis-HCl (pH 7.5), 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA and 1 mM ATP. The reaction was stopped by adding 200 ul Reagent and the released Pi was monitored by a microcolorimetric method after incubated for additional 30 min at room temperature.
4
Chloroplast ATPase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The chloroplast ATPase activity was determined by measuring the amount of inorganic phosphate (Pi) in the reaction by a microcolorimetric method using an ATPase Activity Assay Kit (Sigma-Aldrich). The assay conditions were the same as those described previously (Chen et al. 2016 (link)).
5
Hsp90 ATPase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Hsp90 ATPase activity was monitored by phosphate hydrolysis using ATPase activity assay kit (Sigma Aldrich) according to the manufacturer's instruction. Briefly, Hsp90 inhibitor treated samples incubated for 0.5 hours at 25°C with 4 mM ATP. After incubation, the absorbance of colorimetric product was measured at 620 nm using the microplate reader (Bio-Rad, Hercules, CA, USA).
6
Serca1 ATPase Activity Assay
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Serca1 activity was measured in SR membrane fractions or whole‐muscle homogenates using an ATPase activity assay kit (Sigma‐Aldrich). Enriched SR fraction was isolated from GA muscles in accordance with previous reports.7 The procedures were identical except for initial homogenization using glass‐to‐glass homogenizer (10 strokes) and final solubilization in assay buffer (40 mM Tris, 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA, pH 7.5). To extract whole‐muscle homogenates, TA muscles were homogenized in assay buffer using glass‐to‐glass homogenizer (15 strokes). Homogenates were cleared by centrifugation (3000 rpm for 10 min, then 13 000 rpm for 20 min) and quantified using BCA assay. SR membrane fractions (100 ng) or muscle homogenates (500 ng) were incubated with increasing concentrations of CaCl2 and 750 nM A23187 in assay buffer for 10 min. ATP (1 mM final concentration) was added to initiate the reaction and incubated for 30 min, and then the reagent was added and the mixture incubated for 30 min to generate the colorimetric product. Relative Serca1 activity was plotted with added Ca2+ concentration or maximal Serca1 activity was determined.
7
Protein Extraction and ATPase Quantification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed in a protein lysate buffer (50 mM Tris pH 7.4, 100 μM EDTA, 0.25 M sucrose, 1% SDS, 1% NP40, 1 μg/ml leupeptin, 1 μg/ml pepstatin A and 100 μM phenyl methyl sulfonyl flouride) and homogenized. Debris was removed by centrifugation at 10,000 × g for 10 min at 4 °C, and protein concentrations were measured ATPase Activity Assay Kit (Sigma-Aldrich; malachite green assay, MAK113) following the manual provided by the manufacturer. Absorbance was read at 620 nm, and free phosphate was calculated.
8
Measuring SERCA2 ATPase Activity
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ATPase activity of SERCA2 was examined with an ATPase Activity Assay kit (MAK113; Sigma-Aldrich) according to the manufacturer’s instructions using multifunctional microplate reader (BioTek Synergy H1). Briefly, adipose tissue samples (20 mg) or adipocytes (∼1 × 106 cells) were homogenized on ice in with 200 μl of ice-cold assay buffer (40 mM Tris, 80 mM NaCl, 8 mM MgAc2, 1 mM EDTA, pH 7.5). The homogenates were centrifuged at 14,000 rpm for 10 min at 4°C to remove insoluble material. The reaction was performed in the absence or presence of 10 μM TG at 30°C for 30 min. The TG-sensitive Ca2+-ATPase (SERCA2-ATPase) was calculated by subtracting TG insensitive Ca2+-ATPase activity from total activity and normalized to SERCA protein content.
9
Quantitative ATPase Activity Assay
10
Quantifying ATPase Activity Colorimetrically
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ATPase Activity Assay Kit (Sigma-Aldrich, MAK-113) was used to measure ATPase activity. The protein was diluted to a final concentration of 2 μmol/L, incubated with 1 mmol/L ATP in 40 μL reaction mixture which contained 20 mmol/L MES pH 6.5, 150 mmol/L NaCl, 0.06% digitonin, 5 mmol/L MgCl2 for 30 min at 37°C. Next, the enzyme reactions were stopped and the colourimetric product was generated by adding 200 μL of Reagent (MAK-113) into each reaction well. After incubating for an additional 30 min at room temperature, the absorbance at 620 nm in each well was measured. ATPase activity was represented as the amount of phosphate produced from the ATP catalytic reaction. The experiments were performed in triplicate. Results were analysed in GraphPad Prism 5.0.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!