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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Burnett G, Kennedy EP (1954) The enzymatic phosphorylation of proteins. J Biol Chem 211:969–980
Fischer EH, Krebs EG (1955) Conversion of phosphorylase b to phosphorylase a in muscle extracts. J Biol Chem 216:121–132
Brognard J, Hunter T (2011) Protein kinase signaling networks in cancer. Curr Opin Genet Dev 21:4–11
Zhang H, Pelech S (2012) Using protein microarrays to study phosphorylation-mediated signal transduction. Semin Cell Dev Biol 23:872–882
Zhang J, Yang PL, Gray NS (2009) Targeting cancer with small molecule kinase inhibitors. Nat Rev Cancer 9:28–39
Taniguchi CM, Emanuelli B, Kahn CR (2006) Critical nodes in signalling pathways: insights into insulin action. Nat Rev Mol Cell Biol 7:85–96
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 12:289–309
Mayya V, Han DK (2009) Phosphoproteomics by mass spectrometry: insights, implications, applications and limitations. Expert Rev Proteomics 6:605–618
Schulze WX (2010) Proteomics approaches to understand protein phosphorylation in pathway modulation. Curr Opin Plant Biol 13:280–287
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 8:226–231
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):ra3
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 55–80
Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Pelech S, Sutter C, Zhang H (2003) Kinetworks™ protein kinase multiblot analysis. Methods Mol Biol 218:99–111
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Zhang, H., Shi, X., Pelech, S. (2016). Monitoring Protein Kinase Expression and Phosphorylation in Cell Lysates with Antibody Microarrays. In: Zegzouti, H., Goueli, S. (eds) Kinase Screening and Profiling. Methods in Molecular Biology, vol 1360. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3073-9_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3073-9_9
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3072-2
Online ISBN: 978-1-4939-3073-9
eBook Packages: Springer Protocols