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Universal Kinase Activity Kit, 2 Plate

Catalog #: EA004
13 Citations Datasheet / COA / SDS
Measurement of kinase activity
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EA004
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Citations (13)
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Universal Kinase Activity Kit, 2 Plate Summary

View FMAP Assay Principle
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.

Specifications

Source
N/A
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

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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.

13 Citations: Showing 1 - 10
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  1. An Fgr kinase inhibitor attenuates sepsis-associated encephalopathy by ameliorating mitochondrial dysfunction, oxidative stress, and neuroinflammation via the SIRT1/PGC-1 alpha signaling pathway
    Authors: Liu, Y;Yang, H;Luo, N;Fu, Y;Qiu, F;Pan, Z;Li, X;Jian, W;Yang, X;Xue, Q;Luo, Y;Yu, B;Liu, Z;
    Journal of translational medicine  2023-07-20
  2. Compounds activating VCP D1 ATPase enhance both autophagic and proteasomal neurotoxic protein clearance
    Authors: L Wrobel, SM Hill, A Djajadiker, M Fernandez-, C Karabiyik, A Ashkenazi, VJ Barratt, E Stamatakou, A Gunnarsson, T Rasmusson, EW Miele, N Beaton, R Bruderer, Y Feng, L Reiter, MP Castaldi, R Jarvis, K Tan, RW Bürli, DC Rubinsztei
    Nature Communications, 2022-07-16;13(1):4146.  2022-07-16
  3. Overexpression of PpSnRK1alpha in Tomato Increased Autophagy Activity under Low Nutrient Stress
    Authors: J Luo, W Yu, Y Xiao, Y Zhang, F Peng
    International Journal of Molecular Sciences, 2022-05-13;23(10):.  2022-05-13
  4. Yeast-produced fructosamine-3-kinase retains mobility after ex vivo intravitreal injection in human and bovine eyes as determined by Fluorescence Correlation Spectroscopy
    Authors: AK Minnaert, L van Schie, H Grootaert, J Himpe, S Devos, W Weyts, H De Keersma, K Braeckmans, E Van Aken, JR Delanghe, SC De Smedt, N Callewaert, K Remaut
    International journal of pharmaceutics, 2022-04-26;621(0):121772.  2022-04-26
  5. A negative feedback loop of TOR signaling balances growth and stress-response trade-offs in plants
    Authors: M Jamsheer K, S Jindal, M Sharma, P Awasthi, S S, M Sharma, CT Mannully, A Laxmi
    Cell Reports, 2022-04-05;39(1):110631.  2022-04-05
  6. Mechanistic insights into COVID-19 by global analysis of the SARS-CoV-2 3CLpro substrate degradome
    Authors: I Pablos, Y Machado, HCR de Jesus, Y Mohamud, R Kappelhoff, C Lindskog, M Vlok, PA Bell, GS Butler, PM Grin, QT Cao, JP Nguyen, N Solis, S Abbina, W Rut, JC Vederas, L Szekely, A Szakos, M Drag, JN Kizhakkeda, K Mossman, JA Hirota, E Jan, H Luo, A Banerjee, CM Overall
    Cell Reports, 2021-10-09;37(4):109892.  2021-10-09
  7. A Potential Role for Fructosamine-3-Kinase in Cataract Treatment
    Authors: S De Bruyne, L van Schie, J Himpe, F De Somer, I Everaert, W Derave, C Van den Br, M Huizing, N Bostan, M Speeckaert, N Callewaert, E Van Aken, JR Delanghe
    International Journal of Molecular Sciences, 2021-04-07;22(8):.  2021-04-07
  8. NEK9, a novel effector of IL-6/STAT3, regulates metastasis of gastric cancer by targeting ARHGEF2 phosphorylation
    Authors: G Lu, S Tian, Y Sun, J Dong, N Wang, J Zeng, Y Nie, K Wu, Y Han, B Feng, Y Shang
    Theranostics, 2021-01-01;11(5):2460-2474.  2021-01-01
  9. Discovery of a novel small-molecule inhibitor of Fam20C that induces apoptosis and inhibits migration in triple negative breast cancer
    Authors: R Zhao, L Fu, Z Yuan, Y Liu, K Zhang, Y Chen, L Wang, D Sun, L Chen, B Liu, L Zhang
    European Journal of Medicinal Chemistry, 2020-12-07;210(0):113088.  2020-12-07
  10. 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., 2020-03-23;0(0):.  2020-03-23
  11. 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., 2019-09-26;51(9):112.  2019-09-26
  12. 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, 2017-09-07;2(17):.  2017-09-07
  13. 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, 2015-01-20;0(0):.  2015-01-20

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.

  2. Does the Universal Kinase Activity Kit, 2 Plate (Catalog # EA004) include a kinase and an acceptor substrate?

    • The Universal Kinase Activity Kit is designed to be flexible to work with a variety of kinases and acceptor substrates. The kinase and acceptor substrates need to be supplied by the end user.

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