Universal Kinase Activity Kit, 2 Plate

Measurement of kinase activity
Catalog # Availability Size / Price Qty
EA004
Product Details
Citations (4)
FAQs
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Universal Kinase Activity Kit, 2 Plate Summary

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View Universal Kinase Activity Assay Principle
The Universal Kinase Activity Kit (Catalog # EA004) provides a simple, non-radioactive, high-throughput compatible format for assaying the enzyme activity of kinases in vitro. The majority of kinases use ATP as the phosphate donor. They transfer the terminal phosphate group of ATP to a substrate, producing ADP as a by-product. This kit is an ADP-based phosphatase-coupled kinase assay that utilizes CD39L2/ENTPD6 as a coupling phosphatase. CD39L2 releases the beta-phosphate from ADP, and the released inorganic phosphate is detected using malachite green reagents. The amount of inorganic phosphate released is proportional to the amount of ADP generated during the kinase reaction and thus, reflects the kinetics of the reaction.

 

Features of the Universal Kinase Activity Kit

Versatile

  • Applicable for all kinase reactions that produce ADP

Convenient

  • Non-radioactive
  • No time-consuming separation steps (e.g. column chromatography)

Quantitative

  • 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
 

Kit Contents

  • CHO cell-expressed Recombinant Mouse CD39L2/ENTPD6
  • ATP
  • ADP
  • 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.

 

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.

Data Example

Activity of PKA C beta

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.

Specifications

Shipping Conditions
The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.
Storage
Store the unopened product at -20 to -70 °C. Use a manual defrost freezer and avoid repeated freeze-thaw cycles. Do not use past expiration date.
Species
Multi-Species

Product Datasheets

Citations for Universal Kinase Activity Kit, 2 Plate

R&D Systems personnel manually curate a database that contains references using R&D Systems products. The data collected includes not only links to publications in PubMed, but also provides information about sample types, species, and experimental conditions.

4 Citations: Showing 1 - 4
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  1. 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
  2. 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
  3. 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
  4. 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

FAQs

  1. 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.

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