PP2A activity was performed as previously described using the malachite green assay kit (Millipore-Sigma, Burlington, MA)(25 (link)). The lysates or immunoprecipitates were washed with phosphatase assay buffer and subjected to PP2A activity assay by addition of PP2A specific threonine phospho-peptide substrate. In addition to using commercially available PP2A phospho-peptide substrate, we also generated in-house the threonine phosphopeptide used for PP2A activity assays, described in the supplementary methods .
Malachite green assay kit
Manufactured by Merck Group
Sourced in United States, Switzerland
The Malachite green assay kit is a laboratory tool designed to quantify inorganic phosphate release. It provides a colorimetric method for the detection and measurement of phosphate concentrations in various biological samples.
Automatically generated - may contain errors
6 protocols using malachite green assay kit
1
PP2A Activity Assay Protocol
2
PP2A Activity Measurement Using Malachite Green
3
Malachite Green Assay for PTP1B
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All assays were performed using 2 nM WT PTP1B and a Malachite green assay kit (Sigma Aldrich) in TBS, 2 mM TCEP and 0.01 mg/ml BSA in 96-well plates. 80 μL reactions were set up with peptide at varying concentrations and reactions were started by the addition of PTP1B. After incubation times, reactions were quenched by the addition of working reagent provided in the assay kit. Plates were read using a chameleon V plate reader at 620 nm and results were analyzed using Prism31 . For all assays, two technical replicates were used and a peptide minus PTP1B was measured to determine the amount of free phosphate already in solution, and this was subtracted from the amount measured in the reactions.
4
Quantification of Ectonucleotidase Activity
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Ectonucleotidase activity was measured in the cells as described previously [32 (link)]. Briefly, cells were seeded at 10,000 cells/cm2 in 24-well plates and then incubated for 24 h. ATP, ADP and AMP (1 mM; Santa Cruz) were then added in phosphate-free buffer and incubated for either 1 h (ATP and ADP) or 30 min (AMP). Supernatant was harvested for protein quantification (BCA assay; Thermo Fisher, Waltham, MA, USA), inorganic phosphate quantification (malachite green assay kit, according to the manufacturer’s instructions; Sigma Aldrich) or adenosine detection.
5
Colorimetric Assay for ATP Hydrolysis
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Colorimetric determination of Pi produced by ATP hydrolysis was performed using the Malachite Green Assay Kit (Sigma-Aldrich, Switzerland) and as described previously.49 (link) Several concentrations of Hsp70 (SSA1) and JDPs (YY, SS, SY, YS) were mixed with or without substrate (200 nM of preaggregated luciferase) and with 1 mM of ATP and incubated for 1 h at 25 °C. Exactly 4 µL of each sample was taken and put inside a 96-well plate with 76 µL of H2O. A 20-µL volume of Malachite Green reaction buffer was added, and the samples were mixed thoroughly and incubated at 25 °C for 30 min before measuring at 620 nm on a plate reader (HIDEX-Sense 425–301, Finland). The rate of intrinsic ATP hydrolysis was deduced by subtracting the signal from ATP in the absence of a chaperone.
6
ATP Hydrolysis Assay Protocol
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The ATP hydrolysis reaction was performed as follows. Enzyme was incubated in the assay buffer (50 mM Tris-HCl [pH 7.5], 125 mM KCl, 1 mM EDTA, 5 mM MgCl2, 5 mM DTT, 10% glycerol) with test compounds at various concentrations, 1 mM ATP, and 10.5 nM linear pBR322 DNA for 30 min at room temperature. The protein concentrations used in the assay were as follows: 1 μM PfB, 0.5 μM EcA2PfB2, and 100 nM EcGyr. A malachite green assay kit (Sigma-Aldrich) was used to measure the released phosphate in the reaction. In brief, malachite green reagent was added to the reaction mixture, and the mixture was further incubated for 10 min. The formed color was measured at 620 nm using a plate reader (SpectraMAX190; Molecular Devices). The amount of released phosphate was calculated based on a calibration curve covering a linear range of 50 to 250 pmol using eight concentrations, which was prepared for each measurement independently. Experiments were carried out in triplicate.
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