GST-MoPdeH, His-MoImd4, His-T268AR269A, His-D380AG382A, His-R458AY459A, His-G403A, His-Y428A, His-D290A, His-G340AG342A, His-M431AG432A, His-Q470A and His were induced and purified as described before (Yang et al., 2017 (link)). The phosphodiesterase activity was detected by fluorescence intensity. The MDCC fluorophore coupled with phosphate and the fluorescence of Phosphate Sensor increases approximately 6- to 8-fold which can be measured in real time (Brune et al., 1994 (link)). The reaction system was: 5 cM cAMP (Meilune P/N MB3159), purified GST-MoPdeH with or without equal purified His-fusion proteins, separately, 10 mU/mL alkaline phosphatase (Calbiochem P/N 524545) were incubated in PDE enzymatic reaction buffer for 60 min at room temperature (PDE enzymatic reaction buffer: 50 mM Tris pH 7.6, 100 mM NaCl, 10 mM MgCl2, 0.01% Triton® X-100, and 0.5 mM DTT), then added 0.5 μM Phosphate Sensor (Invitrogen P/N PV4406) in Phosphate Sensor detection buffer to each well (Phosphate Sensor detection buffer: 20 mM Tris pH 7.6 and 0.05% Triton® X-100). The plate was mixed and read by a 10-min kinetic reaction at excitation 420 nm and emission 450 nm. Positive and negative controls were included as previously described (Yang et al., 2017 (link)). The experiments were repeated three times and each experiment had three replicates.
Phosphate sensor
Manufactured by Thermo Fisher Scientific
The Phosphate Sensor is a laboratory instrument designed to measure the concentration of phosphate ions in a sample. It provides accurate and reliable phosphate measurements for various applications, including water quality analysis, environmental monitoring, and process control.
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Lab products found in correlation
Synergy 2 multi mode microplate reader, by Agilent Technologies (1 mentions)
Parp1, by Thermo Fisher Scientific (1 mentions)
Spark 10m, by Tecan (1 mentions)
Adenosine triphosphate (atp), by New England Biolabs (1 mentions)
Low volume non binding surface 384 well plates, by Corning (1 mentions)
Envision plate reader, by PerkinElmer (1 mentions)
5 protocols using phosphate sensor
1
Phosphodiesterase Activity Assay Protocol
2
Monitoring GTPase Activity of Septin Complex
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A 4-μg amount of recombinant septin complex (wild type or mutant) was diluted in 20 μl of GTPase buffer with the addition of phosphate sensor (Invitrogen, Carlsbad, CA) to a concentration of 2 μM. The reaction was started with the addition of GTP to a concentration of 100 μM. phosphate sensor is a phosphate-binding protein modified with a fluorophore. As the sensor binds free Pi, fluorescence intensity increases. The measurement was taken with a Synergy 2 multimode microplate reader (Biotek, Winooski, VT) at excitation of 360(40) nm and emission of 460(40) nm.
3
Quantifying RNF213 ATPase Activity
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RNF213 proteins fused with EGFP were expressed in HEK293 cells, and cells were extracted with RIPA buffer. The cell extract was clarified by high-speed centrifugation and used for immunoprecipitation with anti-GFP agarose (MBL Japan, Nagoya, Japan). Immunoprecipitants were washed with RIPA buffer twice and with RIPA buffer without SDS once, and finally equilibrated with 0.5× kinase buffer (10 mmol/L of Tris-HCl [pH 7.6], 100 mmol/L of KCl, 5 mmol/L of MgCl2, and 0.025% TritonX-100). Immunoprecipitants were resuspended into 50 μL of kinase buffer, and half of this volume was subjected to SDS-PAGE and stained with GelCode staining (Thermo Scientific). To perform ATPase reactions, the indicated volume of immunoprecipitants was combined with prewarmed 1× kinase buffer with 60 μmol/L of ATP. The 50-μL ATPase reaction proceeded for 30 minutes at room temperature by adding a final concentration of Phosphate Sensor (0.5 μmol/L; Thermo Scientific). The plate was mixed and immediately read on a microplate reader at an excitation of 430 nm and emission of 450 nm.
4
Fluorescence-based ATPase Activity Assay
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ATPase activity was measured as described previously (Ahel et al., 2009 (link)) by monitoring the fluorescence intensity of a phosphate binding protein (Brune et al., 1994 (link), Ferreira et al., 2007 (link)) labeled with a coumarin-based fluorescent dye, 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl) coumarin (MDCC), as a readout of the amount of inorganic phosphate (Pi) generated by ATP hydrolysis. Upon binding Pi, the fluorescence of the labeled phosphate binding protein (MDCC-PBP) increases and its emission wavelength shifts. The increase in fluorescence was recorded in solution using a Spark 10 M (Tecan) instrument. 25 μL ATPase reactions were carried out in 50 mM Tris pH 7.9, 50 mM NaCl, and 5 mM MgCl2 buffer with 80 nM ALC1 and 1 mM ATP. Where indicated, 50 μM NAD+ and 80 nM PARP1 (Thermo Fisher) and/or 120 nM 239 bp dsDNA were added. Upon incubation for 2 h at 37°C, phosphate sensor (Thermo Fisher) was added at a final concentration of 0.5 μM and immediately measured (excitation = 420 nm, emission = 465 nm).
5
CHD1L Enzyme Inhibition Assay
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