Universal Kinase Activity Kit, 2 Plate
Universal Kinase Activity Kit, 2 Plate Summary
|View Universal Kinase Activity Assay Principle|
Features of the Universal Kinase Activity Kit
- Applicable for all kinase reactions that produce ADP
- No time-consuming separation steps (e.g. column chromatography)
- The phosphatase provided removes phosphate in a quantitative manner allowing for accurate kinetic analysis
Amenable to High-throughput Analysis
- The assay can be performed in standard multi-well microplates
- Utilizes a standard colorimetric plate reader for analysis
- CHO cell-expressed Recombinant Mouse CD39L2/ENTPD6
- Phosphatase Buffer 4
- Phosphate Standard
- Malachite Green Reagents A & B
Coupling Rates for Kinase Reactions
For accurate kinase activity determination, the amount of ADP produced in the kinase reaction needs to be known. It can be determined using the concentration of inorganic phosphate released from ADP and the coupling rate of the kinase reaction. The coupling rate is defined as the percentage of the ADP product that has been converted to the signal of free inorganic phosphate by the coupling reaction. Coupling rates are dependent on the rate constant, reaction volume, and the reaction time. Coupling rates for a 10 minute, 20 minute, and 30 minute kinase reaction performed using varying amounts of ATP and CD39L2 are provided in the tables below.
- Coupling rates for 10 minute kinase reactions
- Coupling rates for 20 minute kinase reactions
- Coupling rates for 30 minute kinase reactions
If conditions of your experiment, such as pH, temperature, NaCl or Ca2+ concentrations, are different from the conditions used for the tables, the coupling rate can be calculated. Please see the protocol in Technical Resources to determine the rate constant and calculate the coupling rate.
For more detailed technical information, please also see: Wu, Z.L. (2011) PLoS ONE 6(8):e23172.
Activity of PKA C beta
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The activity of Recombinant Human PKA C beta (Catalog # 4596-KS) was assayed using the Universal Kinase Activity Kit (Catalog # EA004) with 0.1 μg of CD39L2 in 50 μL for 10 minutes. The corrected optical density was plotted against kinase concentration. The slope of the line was determined to be 0.908 OD/μg. Using a phosphate conversion factor of 3504 pmol/OD (previously determined), the specific activity was calculated to be 318 pmol/min/μg. After correction with the coupling rate 0.475, the activity is 670 pmol/min/μg.
Protein kinases are the largest class of enzymes in the human genome. These enzymes regulate almost all cellular processes by adding phosphate groups to proteins, thereby modifying the activity, localization, and overall function of their targets. Consequently, abnormal activity of kinases underlies many diseases including cancer.
Citations for Universal Kinase Activity Kit, 2 Plate
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5-Ethynyl-2'-deoxycytidine and 5-ethynyl-2'-deoxyuridine are differentially incorporated in cells infected with HSV-1, HCMV, and KSHV viruses
Authors: S Manska, R Octaviano, CC Rossetto
J. Biol. Chem., 2020;0(0):. 2020
ERK-dependent phosphorylation of the linker and substrate-binding domain of HSP70 increases folding activity and cell proliferation
Authors: S Lim, DG Kim, S Kim
Exp. Mol. Med., 2019;51(9):112. 2019
CF airway smooth muscle transcriptome reveals a role for PYK2
Authors: DP Cook, RJ Adam, K Zarei, B Deonovic, MR Stroik, ND Gansemer, DK Meyerholz, KF Au, DA Stoltz
JCI Insight, 2017;2(17):. 2017
Structural insights into mis-regulation of protein kinase A in human tumors.
Authors: Cheung J, Ginter C, Cassidy M, Franklin M, Rudolph M, Robine N, Darnell R, Hendrickson W
Proc Natl Acad Sci U S A, 2015;0(0):. 2015
Does the Malachite Green Phosphate Detection Kit (DY996) detect both pyrophosphate (diphosphate) and free phosphate (monophosphate)?
The Malachite Green Phosphate Detection Kit measures free inorganic phosphate in aqueous solutions. The assay principle is based on complex formation between malachite green molybdate and free phosphate under acidic conditions. Lipid bound, protein-bound, pyrophashate and other condensed phosphates must be hydrolyzed prior to measurement in the malachite green assay.
Phosphatase Activity Assays
Supplemental ELISA Products
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