Skip to main content

Comparison of SPA, FRET, and FP for Kinase Assays

  • Protocol
High Throughput Screening

Part of the book series: Methods in Molecular Biology™ ((MIMB,volume 190))

Abstract

During the last few years, a variety of technologies have been developed for rapid discovery of protein kinase inhibitors from both synthetic small-molecule libraries and natural products (18). Many of high throughput kinase assays have been developed in 96-, 384-, and 1536-well formats using these technologies (911). Development of these technologies allow one to quickly large synthetic compound or natural product libraries in a very short period of time with high sensitivity, accuracy, and reproducibility. Therefore, development of these new technologies has significantly accelerated the process of discovering drug leads for kinases. In this chapter, I will summarize and compare a number of technologies for development of homogeneous, high-throughput kinase assays.

This is a preview of subscription content, log in via an institution to check access.

Access this chapter

Protocol
USD 49.95
Price excludes VAT (USA)
  • Available as PDF
  • Read on any device
  • Instant download
  • Own it forever
eBook
USD 99.00
Price excludes VAT (USA)
  • Available as EPUB and PDF
  • Read on any device
  • Instant download
  • Own it forever

Tax calculation will be finalised at checkout

Purchases are for personal use only

Institutional subscriptions

References

  1. Beveridge M., Park Y. W. Hermes, J. Marenghi, A. Brophy, and G. Santos, A. (2000) Detection of p56(lck) kinase activity using scintillation proximity assay in 384-well format and imaging proximity assay in 384-and 1536-well format. J. Biomol. Screen 5, 205–212.

    Article  PubMed  CAS  Google Scholar 

  2. Braunwalder A. F., Yarwood D. R, Sills M. A., and Lipson K. E. (1996 a) Measurement of the protein tyrosine kinase activity of c-src using time-resolved fluorometry of europium chelates. Anal. Biochem. 238, 159–164.

    Article  PubMed  CAS  Google Scholar 

  3. Braunwalder A. F., Yarwood D. R., Hall T., Missbach M., Lipson K. E., and Sills M. A. (1996 b) A solid-phase assay for the determination of protein tyrosine kinase activity of c-src using scintillating microtitration plates. Anal. Biochem. 234, 23–26.

    Article  CAS  Google Scholar 

  4. Hemmila I. (1999) LANCEtrade mark: homogeneous assay platform for HTS. J. Biomol. Screen. 4, 303–308.

    Article  PubMed  CAS  Google Scholar 

  5. Parker G. J., Law T. L., Lenoch F. J., and Bolger R. E. (2000) Development of high throughput screening assays using fluorescence polarization: nuclear receptor-ligand-binding and kinase/phosphatase assays. J. Biomol. Screen. 5, 77–88.

    Article  PubMed  CAS  Google Scholar 

  6. Wouters F. S. and Bastiaens P. (1999) Fluorescence lifetime imaging of receptor tyrosine kinase activity in cell. Curr. Biol. 9, 1127–1130.

    Article  PubMed  CAS  Google Scholar 

  7. Wu J. J., Yarwood D. R., Chaudhuri B., Muller L., Zurini M., and Sills M. A. (2000) Measurement of Cdk4 kinase activity using an affinity peptide-tagging technology. Comb. Chem. High Throughput Screen 3, 27–36.

    PubMed  CAS  Google Scholar 

  8. Wu J. J., Yarwood D. R., Pham Q., and Sills M. A. (2000) Identification of a high affinity anti-phosphoserine antibody for the development of a homogeneous fluorescence polarization assay of Protein Kinase C. J. Biomol. Screening 5, 23–30.

    Article  CAS  Google Scholar 

  9. Kowski T. J. and Wu J. J. (2000) Fluorescence polarization is a useful technology for reagent reduction in assay miniaturization. Comb. Chem. High Throughput Screen 3, 437–444.

    PubMed  CAS  Google Scholar 

  10. Fowler A., Swift D., Hemsley P., et al. (2000) A 1536 well fluorescence polarization assay for the Ser/Thr kinase JNK-1, Abstract, the Society for Biomolecular Screening 6th Annual Conference and Exhibition, pp. 196.

    Google Scholar 

  11. Storch E., Ragan S., Born T., Chipman S., and Wu J. J. (2000) Evaluation of different microtiter plate readers for the development of FP and HTR-FRET assays in 384-and 1536-well plates. Abstract for the Society for Biomolecular Screening 6th Annual Conference and Exhibition, pp. 246.

    Google Scholar 

  12. Brophy G., Blair J., and Pither R. (1995) p34cdc2 kinase activity determination using the scintillation proximity assay (SPA). Abstract for the 9th International Conference on Second Messengers and Phosphoproteins. Nashville, TN. pp. 274.

    Google Scholar 

  13. Gobel J., Saussy D. L., and Goetz A. S. (1999) Development of scintillationproximity assays for alpha adrenoceptors. J. Pharmacol. Toxicol. Methods 42, 237–244.

    Article  PubMed  CAS  Google Scholar 

  14. Liu J., Feldman P. A., Lippy J. S., Bobkova E., Kurilla M. G., and Chung T. D. (2001) A scintillation proximity assay for RNA detection. Anal. Biochem. 289, 239–245.

    Article  PubMed  CAS  Google Scholar 

  15. Sherr C. J. (1996) Cancer cell cycles. Science 274, 1672–1677.

    Article  PubMed  CAS  Google Scholar 

  16. Matsushime H., Quelle D. E., Shurtleff S. A., Shibuya M., Sherr C. J., and Kato J. Y. (1994) D-type cyclin-dependent kinase activity in mammalian cells. Mol. Cell. Biol. 14, 2066–2076.

    PubMed  CAS  Google Scholar 

  17. Kitagawa M., Higashi H., Jung H. K., Suzuki-Takahashi I., Ikeda M., Tamai K., et al. (1996) The consensus motif for phosphorylation by cyclin D1-Cdk4 is different from that for phosphorylation by cyclin A/E-Cdk2. EMBO J. 15, 7060–7069.

    PubMed  CAS  Google Scholar 

  18. Schmidt T. G. and Skerra, A. (1993) The random peptide library-assisted engineering of a C-terminal affinity peptide, useful for the detection and purification of a functional Ig Fv fragment. Protein Eng. 6, 109–122.

    Article  PubMed  CAS  Google Scholar 

  19. Stenroos K., Hurskainen P., Eriksson S., Hemmila I., Blomberg K., and Lindqvist C. (1998) Homogeneous time-resolved IL-2-IL-2R alpha assay using fluorescence resonance energy transfer. Cytokine 10, 495–499.

    Article  PubMed  CAS  Google Scholar 

  20. Lynch A. B., Loiacono K. A., Tiong C. L., Adams S. E., and MacNeil I. A. (1997) A fluorescence polarization based Src-SH2 binding assay. Anal. Biochem. 247, 77–82.

    Article  PubMed  CAS  Google Scholar 

  21. Wu P., Brasseur M., and Schindler U. (1997) A high-throughput STAT binding assay using fluorescence polarization. Anal. Biochem. 249, 29–36.

    Article  PubMed  CAS  Google Scholar 

  22. Meijer L., Borgne A., Mulner O., Chong J. P., Blow J., Inagaki N., et al. (1997) Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur. J. Biochem. 243, 527–536.

    Article  PubMed  CAS  Google Scholar 

  23. Phelps D. E. and Xiong Y. (1997) Assay for activity of mammalian cyclin D-dependent kinases CDK4 and CDK6. Methods Enzymol. 283, 194–205.

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

Authors

Editor information

Editors and Affiliations

Rights and permissions

Reprints and permissions

Copyright information

© 2002 Humana Press Inc.

About this protocol

Cite this protocol

Wu, J.J. (2002). Comparison of SPA, FRET, and FP for Kinase Assays. In: Janzen, W.P. (eds) High Throughput Screening. Methods in Molecular Biology™, vol 190. Humana Press. https://doi.org/10.1385/1-59259-180-9:065

Download citation

  • DOI: https://doi.org/10.1385/1-59259-180-9:065

  • Publisher Name: Humana Press

  • Print ISBN: 978-0-89603-889-9

  • Online ISBN: 978-1-59259-180-0

  • eBook Packages: Springer Protocols

Publish with us

Policies and ethics