Kinase‐Catalyzed Biotinylation of Peptides, Proteins, and Lysates

Chamara Senevirathne1, Keith D. Green1, Mary Kay H. Pflum1

1 Department of Chemistry, Wayne State University, Detroit, Michigan
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch110228
Online Posting Date:  March, 2012
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Abstract

Kinase‐catalyzed protein phosphorylation plays an essential role in a variety of biological processes. Methods to detect phosphoproteins and phosphopeptides in cellular mixtures will aid in cell biological and signaling research. Our laboratory recently discovered the utility of γ‐modified ATP analogues as tools for studying phosphorylation. Specifically, ATP‐biotin can be used for labeling and visualizing phosphoproteins from cell lysates. Because the biotin tag is suitable for protein detection, the biotinylation reaction can be applied to multiple phosphoproteomics applications. Herein, we report a general protocol for labeling phosphopeptides and phosphoproteins in biological samples using kinase‐catalyzed biotinylation. Curr. Protoc. Chem. Biol. 4:83‐100 © 2012 by John Wiley & Sons, Inc.

Keywords: kinase; ATP‐biotin; biotin; phosphoprotein; protein labeling

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Kinase‐Catalyzed Biotinylation with Peptide Substrates
  • Basic Protocol 2: Kinase‐Catalyzed Biotinylation with a Full‐Length Protein Substrate
  • Basic Protocol 3: Kinase‐Catalyzed Biotinylation Using Cell Lysates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Kinase‐Catalyzed Biotinylation with Peptide Substrates

  Materials
  • ATP‐biotin (adenosine 5′‐triphosphate [γ]‐biotinyl‐3, 6, 9‐trioxaundecanediamine; Affinity Labeling Technologies, cat. no. 864538‐90‐9; 10 mM stored in methanol)
  • Peptide substrate stock solution (see recipe)
  • Kinase reaction buffer (for PKA and CK2, see recipe)
  • Deionized, purified water
  • ATP [adenosine 5′‐triphosphate; MP Biomedicals, cat. no. ICN15026605: create a 10 mM stock solution in either water or Tris buffer (see recipe)]
  • Kinase enzyme stock solution (see recipe)
  • Acetyl chloride (Acros Organics, cat. no. 75‐36‐5)
  • d 0‐methanol (Acros Organic, cat. no. 67‐56‐1)
  • d 4‐methanol (Cambridge Isotope Labs, cat. no. 811‐98‐3)
  • Matrix solution (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • ThermoSavant SpeedVac (e.g., Model number SPD131DDA)
  • 30°C incubator
  • MALDI plate (standard 384 MTP, Bruker)
  • MALDI‐TOF MS instrument

Basic Protocol 2: Kinase‐Catalyzed Biotinylation with a Full‐Length Protein Substrate

  Materials
  • ATP‐biotin (adenosine 5′‐triphosphate [γ]‐biotinyl‐3, 6, 9‐trioxaundecanediamine; Affinity Labeling Technologies, cat. no. 864538‐90‐9; 10 mM stored in methanol)
  • Protein substrate stock solution (see recipe)
  • Kinase reaction buffer (for CK2, see recipe)
  • Deionized, purified water
  • ATP [adenosine 5′‐triphosphate; MP Biomedicals, cat. no. ICN15026605: create a 10 mM stock solution in either water or Tris buffer (see recipe)]
  • Kinase enzyme stock solution (see recipe)
  • 2× SDS loading buffer (see recipe)
  • 12% or 16% SDS‐polyacrylamide gels (see Sambrook and Russel, )
  • Coomassie Blue stain (NuSep, cat. no. SG021)
  • PVDF membrane (Immobilon PSQ;Millipore, cat. no. IPVH0010
  • Nonfat dry milk
  • PBST buffer (see recipe)
  • Streptavidin‐horseradish peroxidase (SA‐HRP; Pierce, cat. no. EN‐N200)
  • ECL Plus (Amersham Biosciences)
  • ThermoSavant SpeedVac (e.g., Model number SPD131DDA)
  • 30°C incubator
  • Mini‐Protean electrophoretic system (BioRad)
  • Mini‐Transblot cell (BioRad)
  • Gel imaging instrument (e.g., Molecular Dynamics Storm 8600)
  • Additional reagents and equipment for SDS‐PAGE (Sambrook and Russell, )

Basic Protocol 3: Kinase‐Catalyzed Biotinylation Using Cell Lysates

  Materials
  • ATP‐biotin (adenosine 5′‐triphosphate [γ]‐biotinyl‐3, 6, 9‐trioxaundecanediamine, Affinity Labeling Technologies, cat. no. 864538‐90‐9; 10 mM stored in methanol)
  • Kinase reaction buffer (see recipe)
  • Deionized, purified water
  • Cells lysate stock solution (see recipe)
  • SDS loading buffer (see recipe)
  • SpeedVac evaporator
  • 30°C incubator
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Figures

Videos

Literature Cited

Literature Cited
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