CytoPhos Endpoint Phosphate Assay (BK054)

CytoPhos Endpoint Phosphate Assay (BK054)
$0.00

Product Uses Include

  • To measure phosphate in a biological solution or buffer
  • To measure ATP or GTP hydrolysis from an enzyme reaction
  • To measure phosphate release from phosphatase catalyzed reactions

Introduction
The Cytophos™ phosphate assay Biochem Kit™ is an extremely quick and economical way to measure phosphate in solution. There is minimal interference from other common ions and reagents used in molecular biology and biochemistry. The manual is designed to guide you through the process of measuring end-point and kinetic assays. There is special consideration for ATPase and GTPase assays, which can be performed easily with this kit.

The CytoPhos™ phosphate assay is specifically developed for measuring phosphate in lower protein concentration solutions (<0.5 mg/ml). For measuring phosphate in high protein concentration solutions, Cytoskeleton, Inc. recommends using the PhosFree™ phosphate assay (Cat. # BK050).

Kit contents
The kit contains sufficient materials for 1000 assays. The following components are included:

  1. CytoPhos™ reagent
  2. Phosphate standard
  3. Manual with detailed protocols and extensive troubleshooting guide

Equipment needed

  1. Spectrophotometer capable of measuring absorbance at 650 nm wavelength.
  2. Small capacity (100-1000 µl) cuvettes or 96-well microtiter plates.

For product Datasheets and MSDSs please click on the PDF links below.   For additional information, click on the FAQs tab above or contact our Technical Support department at tservice@cytoskeleton.com

AuthorTitleJournalYearArticle Link
Peselj, Carlotta et al.Sterol Metabolism Differentially Contributes to Maintenance and Exit of QuiescenceFrontiers in Cell and Developmental Biology2022ISSN 2296-634X
Duncan, Emily D. et al.Ubiquitylation by Rab40b/Cul5 regulates Rap2 localization and activity during cell migrationThe Journal of cell biology2022ISSN 1540-8140
Jewett, Cayla E. et al.RAB19 Directs Cortical Remodeling and Membrane Growth for Primary CiliogenesisDevelopmental Cell2021ISSN 1878-1551
Goldblum, Rebecca R. et al.Oxidative stress pathogenically remodels the cardiac myocyte cytoskeleton via structural alterations to the microtubule latticeDevelopmental Cell2021ISSN 1878-1551
Boggu, Pulla Reddy et al.Identification of diphenylalkylisoxazol-5-amine scaffold as novel activator of cardiac myosinBioorganic and Medicinal Chemistry2020ISSN 1464-3391
Manickam, Manoj et al.Design and synthesis of sulfonamidophenylethylamides as novel cardiac myosin activatorBioorganic and Medicinal Chemistry2019ISSN 1464-3391
Reidenbach, Andrew G. et al.Conserved Lipid and Small-Molecule Modulation of COQ8 Reveals Regulation of the Ancient Kinase-like UbiB FamilyCell Chemical Biology2018ISSN 2451-9448
Portran, Didier et al.Tubulin acetylation protects long-lived microtubules against mechanical ageingNature Cell Biology2017ISSN 1476-4679
Manickam, Manoj et al.Exploration of flexible phenylpropylurea scaffold as novel cardiac myosin activators for the treatment of systolic heart failureEuropean Journal of Medicinal Chemistry2017ISSN 1768-3254
Matsuyama, Tomonori et al.Midaz(olam) inhibits the hypoxia-induced up-regulation of erythropoietin in the central nervous systemEuropean Journal of Pharmacology2015ISSN 1879-0712
Funk, C. Joel et al.Development of high-throughput screens for discovery of kinesin adenosine triphosphatase modulatorsAnalytical Biochemistry2004ISSN 0003-2697

Question 1:  What is the sensitivity of the CytoPhos Phosphate Assay Biochem Kit (Cat. # BK054)?

Answer 1:  The ATPase assay is sensitive over a range of 1 μM – 50 μM Pi (equivalent to 0.1 nmoles – 5 nmoles Pi in 100 μl reaction volume).

 

 

Question 2:  If assay optimization is necessary with the CytoPhos Phosphate Assay Biochem Kit (Cat. # BK054), what should I focus on?

Answer 2:  The ATPase end-point assay kit has been developed to provide a good general substrate for a broad range of ATPase proteins; however, there are several parameters that may affect ATPase activity, and these include:

 

Protein concentration. A titration of the ATPase of interest should be performed to achieve optimal results. This assay is suitable for protein concentrations below 1 mg/ml. If higher protein concentrations are required, the PhosFree Phosphate Assay Kit (Cat. # BK050) should be considered.

Reaction buffer conditions. Salt concentration (e.g., 20 mM-1000 mM) and pH should be titrated for optimal activity.

ATP concentration. To minimize background readings, an ATP concentration of 0.3 - 0.6 mM is recommended. An ATP titration should be performed to obtain optimal results.

Control Reactions. It is important to include control reactions in the assay, particularly if the ATPase of interest is in an impure state.

Half Area Well Plates. We recommend the use of a half area well plate (180 μl volume) to perform the assays, as this will maintain the pathlength while allowing smaller reaction volumes to be used. The protocols for this assay will describe reactions for a 100 μl final volume. If standard 300 μl volume wells are to be used, we recommend using a 200 μl final volume.