title: A high-density immunoblotting methodology for quantification of total protein levels and phosphorylation modifications.
abstract: The components of many signaling pathways have been identified and there is now a need to conduct quantitative data-rich temporal experiments for systems biology and modeling approaches to better understand pathway dynamics and regulation. Here we present a modified Western blotting method that allows the rapid and reproducible quantification and analysis of hundreds of data points per day on proteins and their phosphorylation state at individual sites. The approach is of particular use where samples show a high degree of sample-to-sample variability such as primary cells from multiple donors. We present a case study on the analysis of >800 phosphorylation data points from three phosphorylation sites in three signaling proteins over multiple time points from platelets isolated from ten donors, demonstrating the technique's potential to determine kinetic and regulatory information from limited cell numbers and to investigate signaling variation within a population. We envisage the approach being of use in the analysis of many cellular processes such as signaling pathway dynamics to identify regulatory feedback loops and the investigation of potential drug/inhibitor responses, using primary cells and tissues, to generate information about how a cell's physiological state changes over time.
authors.affiliation: The University of Reading, Whiteknights, Reading, RG6 6AS, UK.
authors.forename: F
authors.initials: F
authors.lastname: Mazet
authors.affiliation: The University of Reading, Whiteknights, Reading, RG6 6AS, UK.
authors.forename: J L
authors.initials: JL
authors.lastname: Dunster
authors.affiliation: The University of Reading, Whiteknights, Reading, RG6 6AS, UK.
authors.forename: C I
authors.initials: CI
authors.lastname: Jones
authors.affiliation: The University of Reading, Whiteknights, Reading, RG6 6AS, UK.
authors.forename: S
authors.initials: S
authors.lastname: Vaiyapuri
authors.affiliation: The University of Reading, Whiteknights, Reading, RG6 6AS, UK.
authors.forename: M J
authors.initials: MJ
authors.lastname: Tindall
authors.affiliation: The University of Reading, Whiteknights, Reading, RG6 6AS, UK.
authors.forename: M J
authors.initials: MJ
authors.lastname: Fry
authors.affiliation: The University of Reading, Whiteknights, Reading, RG6 6AS, UK.
authors.forename: J M
authors.initials: JM
authors.lastname: Gibbins
mesh_terms: D001792:Blood Platelets / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D015153:Blotting, Western / Q000295:instrumentation / Q000379:methods*; D002352:Carrier Proteins / Q000494:pharmacology; D006801:Humans; D047468:Immunoprecipitation; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism*; D010455:Peptides / Q000494:pharmacology; D051966:Phospholipase C gamma / Q000378:metabolism*; D010766:Phosphorylation; D015539:Platelet Activation / Q000187:drug effects; D061251:Primary Cell Culture; D011499:Protein Processing, Post-Translational; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl / Q000378:metabolism*; D000072377:Syk Kinase
chemical_list: D002352:Carrier Proteins; D047908:Intracellular Signaling Peptides and Proteins; D010455:Peptides; C435908:collagen-related peptide; D050721:Proto-Oncogene Proteins c-cbl; D011505:Protein-Tyrosine Kinases; C000606896:SYK protein, human; D000072377:Syk Kinase; D051966:Phospholipase C gamma; C067419:CBL protein, human