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Radioactive In Vitro Kinase Assays

Overview


Radioactive in vitro kinase assays using purified (or extracted) substrates and kinase utilize 32P-labeled ATP or 35S-thio-labeled ATP (all labeled on the gamma phosphate) to transfer a radioactive phosphate group from ATP to a substrate. Virtually any type of substrate can be used in these assays (protein, peptide, lipid, etc.), as long as a suitable method for separation of radiolabeled ATP and substrate is available. Standard low-throughput formats (when you have just a few samples to test) for radioactive in vitro kinase assays typically use gel electrophoresis, chromatography or filtration techniques to separate radiolabeled ATP from radiolabeled substrate. The amount of phosphorylated substrate is then quantified using standard autoradiography, phosphorimaging, or liquid scintillation counting techniques as appropriate.

What Do I Need To Run This Assay?


  • 32P- gamma-ATP or 35S-gamma-thio-ATP (refer to table in next section)
  • Substrate-of-interest
  • Kinase
  • Chromatography column/vials/scintillation cocktail, or filters/vials/scintillation cocktail, or electrophoresis reagents/equipment and autoradiography film/phosphorimager, or flow scintillation cocktail (to be used with a flow scintillation analyzer for liquid chromatography) as appropriate
  • Liquid scintillation counter (such as a Tri-Carb® counter), autoradiography film developer, phosphorimager (such as a Cyclone™ phosphorimager), or flow scintillation analyzer (FSA) as appropriate

Products and Catalog Numbers


Radiochemicals for Kinase Assays

  • EasyTides® products contain a dye in the buffer to aid with pipetting, and can be stored at 2-8°C
  • 32P and 35S are all beta emitters. "Gamma" in the table below refers to the position of the phosphate on ATP that is labeled.
  • 35S are lower energy radioisotopes, and will provide lower assay background.
  • Products are separated in the last two columns into those that are provided in lead-free outer containers ("pigs"), and those that are provided in lead-lined pigs.
  • Products with higher specific activity indicate that there is more radioactivity per molecule of ATP. Units are given in Curies per millimole of ATP. As there is only one labeled position possible per molecule of ATP (on the gamma phosphate), values that approach the theoretical maximum specific activity for a given radioisotope (~9120 Ci/mmol for 32P or ~ 1488 Ci/mmol for 35S) indicate a greater proportion of the ATP molecules in the stock vial are labeled with the radioisotope. Specific activity can be decreased by adding cold ATP.
CompoundSpecific Activity (Ci/mmol)Rad. conc. (mCi/mL)Molar Concentration (μM)EasyTides Version Containing Dye in Buffer (Shipped ambient, store at 2-8°C)Frozen Version (Shipped on dry ice, store at -20°C)
ATP,[gamma-32P]    102200 BLU002/NEG002
300051.7BLU502H/NEG502HBLU002H/NEG002H
3000103.3BLU502A/NEG502ABLU002A/NEG002A
6000101.7BLU502Z/NEG502ZBLU002Z/NEG002Z
600015025 NEG035C
GTP,[gamma-32P]6000101.7BLU504Z/NEG504ZBLU004Z/NEG004Z
ATP,[gamma-35S]25-10012.5125-500 NEG027
 125012.510 NEG027H

Autoradiography Enhancers for Film or Phosphorimaging

  • EN3HANCE® Autoradiography Enhancer: use on gels for best band resolution
  • ENLIGHTNING™ Rapid Autoradiography Enhancer: safer alternative to 6NE9701. Use on gels.

Scintillation Cocktails

  • Ultima Gold™: multipurpose liquid scintillation cocktail for aqueous and non-aqueous samples
  • Ultima-Flo™ AF: safer flow detection cocktail designed for use with Flow Scintillation Analyzers; accepts gradients up to 2.0 M ammonium formate at a 1:1 ratio with fast and easy mixing; cocktail of choice when using ammonium format buffers to elute radiolabeled inositol phosphates from HPLC columns
  • Ultima-Flo AP: safer flow detection cocktail designed for use with Flow Scintillation Analyzers; accepts gradients up to 2.0 M ammonium phosphate; high counting efficiency and quencyh resistance for a variety of sample types
  • Ultima-Flo M: safer flow detection cocktail designed for use with Flow Scintillation Analyzers; developed for multipurpose flow counting applications; high sample acceptance for a wide range of dilute HPLC eluents, and methanol and acetonitrile gradients
  • Flo-Scint™ cocktails: classical cocktails for flow scintillation analysis

Detection Instruments

  • Tri-Carb® liquid scintillation counter
  • Cyclone™ phosphorimager
  • TopCount® high-throughput detector for plates
  • MicroBeta® high-throughput detector for plates
  • FSA (flow scintillation analyzer) for flow scintillation analysis

Citations


  1. Carlson, H.K. et al. Use of a semisynthetic epitope to probe histidine kinase activity and regulation. Analytical Biochemistry 397, 139-143 (2010). 
  2. Harrison, B.C. et al. Protein kinase C-related kinase targets nuclear localization signals in a subset of class IIa histone deacetylases. FEBS Lett 584, 1103-1110 (2010). 
  3. Ikebe, M., Hartshorne, D.J. & Elzinga, M. Phosphorylation of the 20,000-dalton light chain of smooth muscle myosin by the calcium-activated, phospholipid-dependent protein kinase. Phosphorylation sites and effects of phosphorylation. Journal of Biological Chemistry 262, 9569 -9573 (1987). 
  4. Janoueix-Lerosey, I. et al. Somatic and germline activating mutations of the ALK kinase receptor in neuroblastoma. Nature 455, 967-970 (2008). 
  5. Olsson, H. et al. 4-Anilino-6-phenyl-quinoline inhibitors of mitogen activated protein kinase-activated protein kinase 2 (MK2). Bioorganic & Medicinal Chemistry Letters 20, 4738-4740 (2010). 

Other PerkinElmer Kinase Assay Technologies


  • LANCE Ultra TR-FRET kinase assays
  • LANCE Classic TR-FRET kinase assays
  • AlphaScreen SureFire no-wash cellular kinase assays
  • AlphaScreen kinase assays
  • DELFIA fluorescence kinase assays

Custom Services at PerkinElmer


PerkinElmer offers custom radiochemicals (including GMP-certified radiochemicals) and other custom services. If you are interested in custom services, please contact our custom teams:

ON>POINT® Custom Assay Development Services