Universal Non‐Antibody Detection of Protein Phosphorylation Using pIMAGO

Anton B. Iliuk1, W. Andy Tao2

1 Tymora Analytical Operations, LLC, West Lafayette, 2 Department of Medicinal Chemistry & Molecular Pharmacology, Purdue University, West Lafayette, Indiana
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch140208
Online Posting Date:  March, 2015
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Abstract

This article describes methods for a new, non‐antibody phosphorylation detection reagent, termed pIMAGO (phospho‐imaging). This novel reagent takes advantage not only of the unique properties of the soluble nanoparticles, but also of the multiple functionalities of the molecule, allowing for highly selective, sensitive, and quantitative assessment of protein phosphorylation without using radioactive isotopes or phospho‐specific antibodies. The methods allow for multiplexed detection of phosphorylation and total protein amount simultaneously. The straightforward and routine detection and quantitation of general phosphorylation on any site of any protein can be performed in western blot and ELISA formats. © 2015 by John Wiley & Sons, Inc.

Keywords: phosphoprotein detection; phosphorylation; ELISA; western blot; kinase assay; high‐throughput screening

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: pIMAGO‐Based Phosphoprotein Detection in Western Blot Format
  • Basic Protocol 2: pIMAGO‐Based Phosphoprotein Detection in ELISA Format
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: pIMAGO‐Based Phosphoprotein Detection in Western Blot Format

  Materials
  • Phosphorylated protein (e.g., β‐casein) as a control
  • 4× SDS sample loading buffer
  • 200 mM dithiothreitol solution (prepare fresh)
  • 400 mM iodoacetamide solution (prepare fresh and protect from light)
  • Blocking buffer for western blot (see recipe)
  • pIMAGO reagent (Tymora Analytical, cat. no. PMGO)
  • pIMAGO buffer (see recipe)
  • Washing buffer (see recipe)
  • Avidin‐peroxidase conjugate, for ECL‐based detection (Sigma, cat. no. A3151)
  • Avidin‐fluorophore conjugate, for fluorescence‐based detection (multiple suppliers with various fluorophores are available)
  • 1× TBST (see recipe)
  • Additional reagents and equipment for SDS‐PAGE (Gallagher, ) and immunoblotting (Gallagher, , Ursitti et al., ).

Basic Protocol 2: pIMAGO‐Based Phosphoprotein Detection in ELISA Format

  Materials
  • Phosphorylated protein (e.g., β‐casein) as a control
  • Carbonate buffer (see recipe)
  • Blocking buffer (see recipe)
  • 1× TBST (see recipe)
  • pIMAGO reagent (Tymora Analytical, cat. no. PMGO)
  • pIMAGO buffer (see recipe)
  • Avidin‐peroxidase conjugate, for colorimetry‐based detection (Sigma, cat. no. A3151)
  • Avidin‐fluorophore conjugate, for fluorescence‐based detection (multiple suppliers with various fluorophores are available)
  • Colorimetric TMB peroxidase substrate kit, for colorimetry‐based detection (Bio‐Rad, cat. no. 172‐1066)
  • 2% Oxalic acid prepared in water (protect from light)
  • 96‐well, clear High Bind polystyrene plate (Sigma, cat. no. CLS3590)
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Figures

Videos

Literature Cited

Literature Cited
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   Boas, U. and Heegaard, P. M. 2004. Dendrimers in drug research. Chem. Soc. Rev. 33:43‐63.
   Gallagher, S. 2001. Immunoblot detection. Curr. Protoc. Prot. Sci. 4:10.10.1‐10.10.12.
   Gallagher, S.R. 2012. One‐dimensional SDS gel electrophoresis of proteins. Curr. Protoc. Prot. Sci. 68:10.1.1‐10.1.44.
   Hunter, T. 2000. Signaling–2000 and beyond. Cell 100:113‐127.
   Iliuk, A. , Martinez, J.S. , Hall, M.C. , and Tao, W.A. 2011. Phosphorylation assay based on multifunctionalized soluble nanopolymer. Anal. Chem. 83:2767‐2774.
   Iliuk, A.B. , Martin, V.A. , Alicie, B.M. , Geahlen, R.L. , and Tao, W.A. 2010. In‐depth analyses of kinase‐dependent tyrosine phosphoproteomes based on metal ion‐functionalized soluble nanopolymers. Mol. Cell Proteomics. 9:2162‐2172.
   Iliuk, A. , Liu, X.S. , Xue, L. , Liu, X. , and Tao, W.A. 2012. Chemical visualization of phosphoproteomes on membrane. Mol. Cell. Proteomics 11:629‐639.
   Jensen, S.S. and Larsen, M.R. 2007. Evaluation of the impact of some experimental procedures on different phosphopeptide enrichment techniques. Rapid. Commun. Mass Spectrom. 21:3635‐3645.
   Larsen, M.R. , Thingholm, T.E. , Jensen, O.N. , Roepstorff, P. , and Jorgensen, T.J. 2005. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumns. Mol. Cell Proteomics. 4:873‐886.
   Nawrocki, J. , Dunlap, C. , McCormick, A. , and Carr, P.W. 2004. Part I. Chromatography using ultra‐stable metal oxide‐based stationary phases for HPLC. J. Chromato. A 1028:1‐30.
   Pawson, T. 2004. Specificity in signal transduction: From phosphotyrosine‐SH2 domain interactions to complex cellular systems. Cell 116:191‐203.
   Torta, F. , Fusi, M. , Casari, C.S. , Bottani, C.E. , and Bachi, A. 2009. Titanium dioxide coated MALDI plate for on target analysis of phosphopeptides. J. Proteome. Res. 8:1932‐1942.
   Ursitti, J.A. , Mozdzanowski, J. , and Speicher, D.W. 2001. Electroblotting from polyacrylamide gels. Curr. Protoc. Prot. Sci. 00:10.7.1‐10.7.14.
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