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Pde5a1 assay kit

Manufactured by BPS Biosciences
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The PDE5A1 Assay kit is a laboratory tool designed to measure the activity of the PDE5A1 enzyme. It provides the essential components to perform the assay, including the PDE5A1 enzyme, substrate, and detection reagents. The kit enables researchers to quantify the enzymatic activity of PDE5A1 in a controlled experimental setting.

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Lab products found in correlation

Envision 2105 multimode plate reader, by PerkinElmer (1 mentions) Deuterated chloroform cdcl3, by Fujifilm (1 mentions) Spectramax paradigm, by Molecular Devices (1 mentions) Softmax pro, by Molecular Devices (1 mentions)

4 protocols using pde5a1 assay kit

1

PDE5 Enzyme Activity Assay

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The PDE5 enzyme activity was determined using PDE5A1 assay kit (BPS Bioscience, San Diego, CA, USA) based on the PDE5 catalytic ability to hydrolyze dye-labeled cyclic monophosphates, which can further be bound by selective beads and lead to slower rotating and lower polarized light emission. The compounds that inhibited PDE5 enzyme demonstrated affected polarized fluorescence. Following the manufacturer’s protocol, after incubating compounds with PDE5A1 enzyme and FAM-Cyclic-3′, 5′-GMP in buffers for 1 h, the fluorescent polarization was detected and calculated.
Chang Y.C., Tseng Y.L., Leu W.J., Du C.M., Jiang Y.H., Hsu L.C., Hsu J.L., Hou D.R, & Guh J.H. (2020). Discovery of Novel Agents on Spindle Assembly Checkpoint to Sensitize Vinorelbine-Induced Mitotic Cell Death against Human Non-Small Cell Lung Cancers. International Journal of Molecular Sciences, 21(16), 5608.
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2

Evaluating PDE5A1 Inhibition by Dipyridamole and TM-dipyridamole

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The PDE5A1 Assay kit (BPS Biosciences) was used to evaluate phosphodiesterase (PDE) inhibition of dipyridamole and TM-dipyridamole according to the manufacturer’s instructions. Briefly, all test compound stocks solutions (500 mM IBMX; 10 mM dipyridamole; 10 mM TM-dipyridamole) were prepared in 100% DMSO and subsequently diluted to 10x working stock concentrations in 10% dimethyl sulfoxide (DMSO) in the PDE assay buffer included in the kit (500 μM IBMX; 30 μM and 100 μM dipyridamole; 30 μM and 100 μM TM-dipyridamole). A final concentration of 1% DMSO was present in each PDE5A1 enzyme inhibition reaction, including the PDE5A1 positive control reaction. Based on preliminary tests, concentrations greater than 1% DMSO negatively-affected the PDE5A1 enzymatic activity (data not shown). The fluorescent polarization of each sample was analyzed using a PerkinElmer EnVision 2105 Multimode Plate Reader equipped with a FITC FP 480 nm excitation filter and emission filters FITC FP P-pol 535 nm and FITC S-pol 535 nm. Milli-polarization values (mP) were calculated with the EnVision Workstation v1.12 software and PDE5A1 enzymatic activity for each reaction was normalized to the enzymatic positive control sample.
Esquejo R.M., Roqueta-Rivera M., Shao W., Phelan P.E., Seneviratne U., am Ende C.W., Hershberger P.M., Machamer C.E., Espenshade P.J, & Osborne T.F. (2020). Dipyridamole inhibits lipogenic gene expression by retaining SCAP-SREBP in the endoplasmic reticulum. Cell chemical biology, 28(2), 169-179.e7.
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3

Sildenafil and Descarbon-sildenafil Assay

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Sildenafil citrate (Product No. S-061) and descarbon- sildenafil (Product No. S-0624) were purchased from TLC Pharmaceutical Standards Ltd (Aurora, Ontario, Canada). 1-Hexanesulfonic acid sodium salt (ion-pair chromatography grade), formic acid in acetonitrile (0.1％ v/v, LC-MS grade) and deuterated chloroform (CDCl 3 ) (99.8％) for NMR analysis were from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan). Dimethyl sulfoxide (DMSO) (Guaranteed Reagent grade) was from Nacalai Tesque Inc (Kyoto, Japan). All other chemicals and solvents were of analytical or HPLC grades. The PDE5A1 Assay Kit was purchased from BPS Bioscience Inc (San Diego, CA, USA).
Ichikawa-Kaji Y., Nakajima J., Kikkawa S., Ishizawa F., Nishiyama R., Kishimoto K., Uemura N., Satoh M., Suzuki J., Inomata A., Nakae D., Azumaya I., Honda K, & Moriyasu T. (2020). [Identification of Descarbonsildenafil in an Adulterated Dietary Supplement and Evaluation of Its Inhibitory Activity for Phosphodiesterase Type 5 (PDE5)]. Shokuhin eiseigaku zasshi. Journal of the Food Hygienic Society of Japan, 61(1).
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4

PDE5A1 Inhibition Assay Protocol

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For the measurement of IC 50 values, the sildenafil citrate standard and the descarbonsildenafil standard were diluted with DMSO to obtain final concentrations from 0.03 to 1,000 nmol/L. In this experiment, the sildenafil citrate standard was prepared as the reference compound. The assay was performed by using a PDE5A1 Assay kit (BPS Bioscience Inc). All tested solu- tions were 10 times diluted with the PDE assay buffer of the kit. The assay protocol and how to calculate fluorescence polarization data were identical to those described elsewhere 7) , except the following points. After the enzymatic reactions, the reaction time with the binding solution was shortened to 30 min (instead of 60 min). Fluo- rescence polarization was measured at an excitation of 485 nm and an emission of 535 nm using SpectraMax Paradigm (Molecular Devices, LLC, Sunnyvale, CA, USA) instead of 485 and 528 nm, respectively. For all the test solutions, the fluorescence polarization was measured in quadruplicate wells. The fluorescence polarization data and the IC 50 value were analyzed using the computer software, SoftMax Pro (Molecular Devic- es).
Ichikawa-Kaji Y., Nakajima J., Kikkawa S., Ishizawa F., Nishiyama R., Kishimoto K., Uemura N., Satoh M., Suzuki J., Inomata A., Nakae D., Azumaya I., Honda K, & Moriyasu T. (2020). [Identification of Descarbonsildenafil in an Adulterated Dietary Supplement and Evaluation of Its Inhibitory Activity for Phosphodiesterase Type 5 (PDE5)]. Shokuhin eiseigaku zasshi. Journal of the Food Hygienic Society of Japan, 61(1).
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