The first and second AAA+ ATPase modules that are fused with GST were purified using GSH beads (GE Healthcare, USA) as previously described14 (link). ATPase activity was measured by malachite green assay using BIOMOL Green kit (Enzo Life Sciences, USA) as previously described14 (link)44 (link).
Biomol green kit
Manufactured by Enzo Life Sciences
Sourced in United States, Germany
The BIOMOL Green kit is a fluorescent detection reagent used for the quantitation of proteins in solution. It provides a sensitive and accurate method for determining protein concentrations in biological samples.
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6 protocols using biomol green kit
1
Purification and Assay of AAA+ ATPase Modules
2
Assay for Na+/K+-ATPase Activity
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Serum‐starved, hormonally defined, treated LLC PK‐1 cells in 24‐well plates were permeabilized by osmotic shock in ultrapure water (150 µL) and fast freeze in liquid nitrogen (10 seconds). Cells were thawed (200 µL) in a buffer with a composition of 200 mmol/L sodium chloride, 80 mmol/L histidine, 20 mmol/L potassium chloride, 6 mmol/L magnesium chloride, 2 mm EGTA, 2 µg/mL Alamethicin, 30 µmol/L Digitonin. For each drug exposure condition, a volume of 30 mmol/L Ouabain (final concentration 1 mmol/L) or ultrapure water was added to every other well. Cells were either incubated 30 minutes at 37°C. A volume of 30 mmol/L ATP (final concentration 1 mmol/L) was added to each well. Cells were incubated 30 minutes more at 37°C. On ice, trichloroacetic acid: water (1:1) terminated ATP hydrolysis reactions. Plates were centrifuged at 1200 g for 10 minutes at room temperature. Supernatants were diluted 50 times and distributed (50 µL) in technical triplicates to a 96‐well plate.
Concentration of free phosphate released from ATP hydrolysis was measured using the BIOMOL® Green kit (BML‐AK‐111‐0250, Enzo). Na+/K+‐ATPase activity (expressed in nmol of released phosphate/30min/105 cells) was calculated as the difference in free phosphate concentration in the presence and absence of Ouabain.
Concentration of free phosphate released from ATP hydrolysis was measured using the BIOMOL® Green kit (BML‐AK‐111‐0250, Enzo). Na+/K+‐ATPase activity (expressed in nmol of released phosphate/30min/105 cells) was calculated as the difference in free phosphate concentration in the presence and absence of Ouabain.
3
Phosphatase Activity Assay for MLCP
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MLCP
activity was determined as the amount
of inorganic phosphate (Pi) released with the phospho-MLC20
peptide as the substrate, unless noted. We used the phosphopeptide
to avoid allosteric effects caused by the multiple-site interaction
with the myosin molecule. The phospho-MLC20 peptide {P-MLC20(3–26)
mimicking human MLC20 residues 3–26 [KRAKAKTTKKRPQRAT(Sp)NVFAMFD]} was synthesized by
LifeTein. The amount of released Pi was measured using
a malachite green assay (BioMol Green kit, Enzo). The assay was performed
in triplicate in a 96-well plate with a final volume of 50 μL.
Conditions included 0.003–0.04 mg/mL recombinant MLCP in the
presence of assay buffer [25 mM MOPS-NaOH (pH 7.0), 0.5 mM TCEP, 0.1
mM EDTA, 4 mM PFB, and 1 nM okadaic acid (OA)] at 30 °C. A small
dose of OA was added to eliminate the activity of potential contaminants
(PP2A/4/6) in the purified MLCP sample. The phosphatase reaction was
initiated by the addition of 5 μL of 0.25 mM P-MLC20(3–26).
After a 30 min incubation, 100 μL of BioMol Green reagent was
added to each well to terminate the reaction. The plate was incubated
at room temperature for 20–30 min, and the absorbance at 650
nm in each well was read by the plate reader. The amount of Pi was obtained using the OD value from the triplicate wells
with a standard Pi solution. The MLCP assay with 32P-labeled MLC20 was conducted as described previously.33 (link)
activity was determined as the amount
of inorganic phosphate (Pi) released with the phospho-MLC20
peptide as the substrate, unless noted. We used the phosphopeptide
to avoid allosteric effects caused by the multiple-site interaction
with the myosin molecule. The phospho-MLC20 peptide {P-MLC20(3–26)
mimicking human MLC20 residues 3–26 [KRAKAKTTKKRPQRAT(Sp)NVFAMFD]} was synthesized by
LifeTein. The amount of released Pi was measured using
a malachite green assay (BioMol Green kit, Enzo). The assay was performed
in triplicate in a 96-well plate with a final volume of 50 μL.
Conditions included 0.003–0.04 mg/mL recombinant MLCP in the
presence of assay buffer [25 mM MOPS-NaOH (pH 7.0), 0.5 mM TCEP, 0.1
mM EDTA, 4 mM PFB, and 1 nM okadaic acid (OA)] at 30 °C. A small
dose of OA was added to eliminate the activity of potential contaminants
(PP2A/4/6) in the purified MLCP sample. The phosphatase reaction was
initiated by the addition of 5 μL of 0.25 mM P-MLC20(3–26).
After a 30 min incubation, 100 μL of BioMol Green reagent was
added to each well to terminate the reaction. The plate was incubated
at room temperature for 20–30 min, and the absorbance at 650
nm in each well was read by the plate reader. The amount of Pi was obtained using the OD value from the triplicate wells
with a standard Pi solution. The MLCP assay with 32P-labeled MLC20 was conducted as described previously.33 (link)
4
PTEN Phosphatase Activity Assay
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The PTEN activity assay was performed was previously described [50] (link). For each reaction, 400 ng recombinant PTEN (rPTEN, R&D, Minneapolis, MN) was incubated with 4-HNE or 4-HHE in 50 mM tricine and 100 mM NaCl (pH = 8.0) assay buffer for 30 min at room temperature. The reaction was then ceased by incubation in 2 mM DTT for 30 min. Next, 40 µM phosphatidylinositol-3,4,5-trisphosphate diC8 (Echelon, Salt Lake City, UT) was applied to 25 µL of the PTEN reaction mixture to detect the phosphatase activity of rPTEN at 37 °C. The reaction was stopped 40 min later. BIOMOL Green kit (Enzo Life Science, Farmingdale, NY) was used to measure the released phosphate. Briefly, 25 µL PTEN reaction samples were incubated with 100 µL of BIOMOL Green for 15 min. The phosphate concentration was measured with a microplate reader (Bio-Rad) at 655 nm. All data were presented as picomoles of phosphate per 25 µL reaction.
5
FtsZ GTPase Activity Quantification
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GTPase activity of FtsZ was measured by quantifying the inorganic phosphate with a colorimetric phosphate quantification assay (BIOMOL GREEN kit, ENZO life sciences, Lörrach, Germany) for 140 s48 (link). Purified FtsZ-wt or FtsZ-G55-Venus-Q56 were used at 3 µM in Reaction buffer, and polymerization was triggered by 1 mM GTP. 13 µL fractions were added to a 96-Well Flat-Bottom Microplate (UV-Star, Greiner Bio-One GmbH) every 20 s after addition of GTP and mixed with 37 µL of Reaction buffer and 100 µL of BIOMOL GREEN reagent, stopping the reaction. After ~10 min of incubation at RT, the absorbance at 620 nm was measured in a TECAN plate reader (Tecan Group Ltd., Mannedorf, Switzerland) at room temperature. Phosphate concentrations were calculated from a Na2HPO4 standard curve in Reaction buffer, and the GTPase activity reaction rate (V, mol Pi/mol FtsZ/min) was determined from the slope of the linear part of phosphate accumulation curves.
6
Quantification of Bacterial Polyphosphate
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Supernatants of lactobacilli strains grown in MEI medium, obtained by centrifugation and filtration, were subjected to phenol and chloroform:isoamyl alcohol (24:1) extraction and the aqueous phases were transferred to fresh tubes. Polyphosphate was precipitated by adding sodium chloride to 0.1 M final concentration and one volume of ethanol 96% (v/v). The tubes were incubated for 3 h at −20°C and centrifuged for 10 min at 13.000 × g at 4°C. The poly-P pellets were washed with 70% cold ethanol and air-dried before being resuspended in 50 μl of water. Poly-P was quantified by fluorescence using 4′,6-diamino-2-fenilindol (DAPI) at a final concentration of 10 μM in 50 mM Tris-HCl pH 7.5, 50 mM NaCl buffer with an excitation wavelength of 415 nm and emission at 550 nm (Aschar-Sobbi et al., 2008 (link)) in a Clariostar fluorimeter (BMG LabTech). Serial dilutions of a sample of poly-P isolated from strain Lpp+ were hydrolyzed with a volume of 2 M HCl and incubation at 95°C 15 min, followed by neutralization by adding half volume of 2 M NaOH. The released phosphate was measured with the BIOMOL Green kit (Enzo Life Sciences) as recommended by the manufacturer. This quantified sample of poly-P was used to build a standard curve for the DAPI method.
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