Monitoring Protein Kinase Expression and Phosphorylation in Cell Lysates with Antibody Microarrays.
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.
Kinexus Bioinformatics Corporation, Suite 1, 8755 Ash Street, Vancouver, British Columbia, Canada, V6P 6T3.
Hong
H
Zhang
Kinexus Bioinformatics Corporation, Suite 1, 8755 Ash Street, Vancouver, British Columbia, Canada, V6P 6T3.
Xiaoqing
X
Shi
Kinexus Bioinformatics Corporation, Suite 1, 8755 Ash Street, Vancouver, British Columbia, Canada, V6P 6T3. spelech@kinexus.ca.|Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada. spelech@kinexus.ca.
Steven
S
Pelech
D000906:Antibodies / Q000276:immunology*; D000918:Antibody Specificity; D000937:Antigen-Antibody Reactions; D002458:Cell Fractionation; D045744:Cell Line, Tumor; D066246:ErbB Receptors / Q000378:metabolism; D006801:Humans; D007091:Image Processing, Computer-Assisted; D007202:Indicators and Reagents; D009363:Neoplasm Proteins / Q000378:metabolism; D010750:Phosphoproteins / Q000276:immunology; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D011494:Protein Kinases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational
D000906:Antibodies; D007202:Indicators and Reagents; D009363:Neoplasm Proteins; D010750:Phosphoproteins; D011494:Protein Kinases; C512478:EGFR protein, human; D066246:ErbB Receptors
Antibody microarray; Phosphoproteomics; Phosphorylation; Protein kinase; Protein kinome