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Monitoring Protein Phosphorylation for the Ligand Screens

Monitoring Protein Phosphorylation for the Ligand Screens. Goal: sample diversity of cellular response to inputs Current Approach: Multiplex Western blotting with mixtures of phosphospecific antibodies Quantify ligand-induced changes in site-specific protein phosphorylation .

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Monitoring Protein Phosphorylation for the Ligand Screens

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  1. Monitoring Protein Phosphorylationfor the Ligand Screens • Goal: sample diversity of cellular response to inputs • Current Approach: • Multiplex Western blotting with mixtures of phosphospecific antibodies • Quantify ligand-induced changes in site-specific protein phosphorylation

  2. Ligand Screens: B Cells • Single ligand screen completed • Two phosphospecific antibody mixes for quantification of 11 phosphoproteins • Timecourse data on-line • Poster: “Analysis of B Cell Single Ligand Data” Robert Sinkovits and Dennis Mock • Dual ligand screen experiments and analysis in progress. See posters: • “Dual Ligand Screen in Splenic B Cells” Keng-Mean Lin, Madhusudan Natarajan, Robert Hsueh, and Heping Han • “Dual Ligand Screen in Splenic B Cells (continued)” Keng-Mean Lin, Madhusudan Natarajan, Robert Hsueh, and Heping Han

  3. Ligand Screens: WEHI-231 • Poised to initiate screen: characterization of cells, derivation of protocols, validation of phosphospecific antibodies completed • See poster: “Preparation and Characterization of WEHI-231 for the Ligand Screens” Robert Hsueh, Keng-Mean Lin, and Heping Han

  4. Ligand Screens: Cardiac Myocytes • Two new antibody mixes developed • Some antibodies that worked well for B cells did not for myocytes • Added antibodies for myocyte specific phosphoproteins • Ligand dose determinations completed • Dual ligand screen initiated. See posters: “Isolation and Culture of Adult Mouse Cardiac Myocytes for Signaling Studies” Timothy D. O'Connell, Yan G. Ni, Keng-Mean Lin, Heping Han, and Zhen Yan “Ligand Screen in Mouse Adult Cardiac Myocytes” Yan G. Ni, Keng-Mean Lin, Heping Han, and Timothy D. O'Connell

  5. Current Phosphospecific Antibody Mixtures Each antibody is directed to a specific site of phosphorylation on protein Color-coding highlights 8 different pathways currently monitored in each cell type Prospective: Can we assay more phosphoproteins? Add ribosomal S6 and myosin light chain to Mix 1 Another mix for leukocytes: Could start with p70 S6 kinase and GSK 3from Mix 3 & 4

  6. Insulin Receptor Signaling * * * * * * * * *Antibody leukocyte mix *Antibody in myocyte mix *Good antibody not in mix yet *

  7. Antibody Database • Tabulation of results from antibody testing by Western immunoblotting • Now available on-line (go to Data Center, Resources) • Easy to spot antibodies that yielded promising results (highlighted with red check marks) • See poster: “AfCS Antibody Laboratory’s Database:Tabulation of Our Experience with Commercially Available Antibodies”Heping Han, Becky Fulin, Lonnie Sorrells, Ruth Levitz, and Susanne Mumby

  8. Monitoring Protein Phosphorylation:Increasing Our Repertoire • 1 of 7 phosphospecific antibodies we test is suitable for multiplex Western blotting of whole cell lysates • Additional promising antibodies to proteins in pathways not represented by antibody Mixes 1 & 2 • Smad 2 (S465/467) • STAT1 (Y701) • PKC delta (T505) • Total current possibilities for multiplex = 10 • Capacity: 80 samples/week with two mixes • Best case scenario • 2 more antibody mixtures for multiplex Western blotting • Doubles the gels+blots, decreases number of samples we can assay to 40/week • Additional antibodies not good enough to multiplex • More cells required • More gels/blots to process means less than 40 samples/week • An alternative, less labor intensive assay could be our salvation

  9. Monitoring More Phosphoproteins:Alternative Assay • Multiplex fluorescent bead technology • Potential for monitoring 100 different analytes per well of 96-well plate • Licensed by Luminex • Bio-Rad’s system: Bio-Plex • Cytokines • Phosphoproteins • AfCS and Bio-Rad collaborate on development and validation of phosphoprotein assays

  10. Bio-Plex Phosphoprotein Assays

  11. Three-way Collaboration 1. AfCS and Cell Signaling Technology • AfCS suggests targets for novel antibodies • CST produces peptide antigen and antibodies • CST and AfCS test antibodies • AfCS uses antibodies and CST sells them 2. CST and Bio-Rad • Collaboration agreement pending • Development of antibody pairs (capture and phosphospecific) for Bio-Plex 3. Bio-Rad and AfCS • Develop/validate antibody pairs for Bio-Plex • Set up Bio-Plex in AfCS to monitor protein phosphorylation

  12. Benefits of 3-way Collaboration • AfCS: • Increase number of phosphoproteins monitored = more data for modeling effort • No increase in number of cells required • CST and Bio-Rad: • Company name and product exposure • Increased sales • Signaling Community: • More AfCS data available to draw from • Increased number of validated antibody reagents available on the market • Demonstration of the utility of Luminex technology for analysis of phosphoproteins

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