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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
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
ligand screens b cells
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

ligand screens wehi 231
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

ligand screens cardiac myocytes
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

current phosphospecific antibody mixtures
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

slide6

Insulin Receptor

Signaling

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*Antibody leukocyte mix

*Antibody in myocyte mix

*Good antibody not in mix yet

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antibody database
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

monitoring protein phosphorylation increasing our repertoire
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
monitoring more phosphoproteins alternative assay
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
three way collaboration
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
benefits of 3 way collaboration
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