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Monitoring Protein Kinase Expression and Phosphorylation in Cell Lysates with Antibody Microarrays

  • Hong Zhang
  • Xiaoqing Shi
  • Steven Pelech
Part of the Methods in Molecular Biology book series (MIMB, volume 1360)

Abstract

Fuelled by advances in our understanding of the human kinome and phosphoproteome and the increasing availability of pan- and phosphosite-specific antibodies, antibody microarrays have emerged as powerful tools for interrogating protein phosphorylation-mediated signaling systems in ex vivo studies. This economical platform permits ultra-sensitive, semiquantitative measurements of the levels of hundreds of protein kinases and their substrates along with their phosphorylation status simultaneously with minute amounts of specimens. Recent technological innovations in the design and fabrication of antibody microarrays and sample preparation have permitted further refinements of the technology to yield improvements in data quality. In this chapter, we describe a detailed protocol that we have developed for tracking the expression and phosphorylation of protein kinases and their substrates in crude cell lysate samples using a high-content antibody microarray.

Key words

Antibody microarray Phosphorylation Phosphoproteomics Protein kinase Protein kinome 

References

  1. 1.
    Burnett G, Kennedy EP (1954) The enzymatic phosphorylation of proteins. J Biol Chem 211:969–980PubMedGoogle Scholar
  2. 2.
    Fischer EH, Krebs EG (1955) Conversion of phosphorylase b to phosphorylase a in muscle extracts. J Biol Chem 216:121–132PubMedGoogle Scholar
  3. 3.
    Brognard J, Hunter T (2011) Protein kinase signaling networks in cancer. Curr Opin Genet Dev 21:4–11PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Zhang H, Pelech S (2012) Using protein microarrays to study phosphorylation-mediated signal transduction. Semin Cell Dev Biol 23:872–882CrossRefPubMedGoogle Scholar
  5. 5.
    Zhang J, Yang PL, Gray NS (2009) Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer 9:28–39CrossRefPubMedGoogle Scholar
  6. 6.
    Taniguchi CM, Emanuelli B, Kahn CR (2006) Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 7:85–96CrossRefPubMedGoogle Scholar
  7. 7.
    Martin L, Latypova X, Wilson CM, Magnaudeix A, Perrin ML, Yardin C, Terro F (2013) Tau protein kinases: involvement in Alzheimer’s disease. Ageing Res Rev 12:289–309CrossRefPubMedGoogle Scholar
  8. 8.
    Mayya V, Han DK (2009) Phosphoproteomics by mass spectrometry: insights, implications, applications and limitations. Expert Rev Proteomics 6:605–618PubMedCentralCrossRefPubMedGoogle Scholar
  9. 9.
    Schulze WX (2010) Proteomics approaches to understand protein phosphorylation in pathway modulation. Curr Opin Plant Biol 13:280–287CrossRefPubMedGoogle Scholar
  10. 10.
    Oyama M, Kozuka-Hata H, Tasaki S, Semba K, Hattori S, Sugano S, Inoue J, Yamamoto T (2009) Temporal perturbation of tyrosine phosphoproteome dynamics reveals the system-wide regulatory networks. Mol Cell Proteomics 8:226–231CrossRefPubMedGoogle Scholar
  11. 11.
    Olsen JV, Vermeulen M, Santamaria A, Kumar C, Miller ML, Jensen LJ, Gnad F, Cox J, Jensen TS, Nigg EA, Brunak S, Mann M (2010) Quantitative phosphoproteomics reveals widespread full phosphorylation site occupancy during mitosis. Sci Signal 3(104):ra3CrossRefPubMedGoogle Scholar
  12. 12.
    Zhang H, Pelech S (2012) Protein microarrays and their potential clinical applications in the era of personalized. In: Jordan B (ed) Microarrays in diagnostics and biomarker development: current and future applications. Springer, Herdelberg, Berlin, pp 55–80CrossRefGoogle Scholar
  13. 13.
    Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  14. 14.
    Pelech S, Sutter C, Zhang H (2003) Kinetworks™ protein kinase multiblot analysis. Methods Mol Biol 218:99–111Google Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Kinexus Bioinformatics CorporationVancouverCanada
  2. 2.Department of MedicineUniversity of British ColumbiaVancouverCanada

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