Fluorescent Peptide Assays for Protein Kinases

Ashwini K. Devkota1, Tamer S. Kaoud1, Mangalika Warthaka1, Kevin N. Dalby1

1 University of Texas at Austin, Austin, Texas
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.17
DOI:  10.1002/0471142727.mb1817s91
Online Posting Date:  July, 2010
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Abstract

Protein kinases are enzymes that regulate many cellular events in eukaryotic cells, such as cell‐cycle progression, transcription, metabolism, and apoptosis. Protein kinases each have a conserved ATP‐binding site, as well as one or more substrate‐binding site(s) that exhibit recognition features for a protein substrate. Thus, by bringing ATP and a substrate into close proximity, each protein kinase can modify its substrate by transferring the γ phosphate of the ATP molecule to a serine, threonine, or tyrosine residue on the substrate. In such a way, signaling pathways downstream from the substrate can be regulated, dependent on the phosphorylated versus dephosphorylated forms of the substrate. This unit describes an assay employing a fluorescent peptide substrate to measure the incorporation of non‐radiolabeled phosphate. The assay is based on the principle that the phosphorylation of the peptide substrate leads to an increase in the fluorescence emission intensity of an appended fluorophore. Curr. Protoc. Mol. Biol. 91:18.17.1‐18.17.7. © 2010 by John Wiley & Sons, Inc.

Keywords: peptide assays; kinases; fluorescent peptides

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Measuring Protein Kinase Activity Using Fluorescent Peptides
  • Alternate Protocol 1: Measuring Protein Kinase Activity Using a Fluorescence Plate Reader
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Measuring Protein Kinase Activity Using Fluorescent Peptides

  Materials
  • 5× assay buffer, pH 7.5 (see recipe)
  • 10 mg/ml bovine serum albumin (BSA; see recipe)
  • 5× (5 mM) non‐radiolabeled ATP (see recipe)
  • 5× (250 µM) Sox‐based peptide (see recipe; also see for a description of available peptides)
  • 10 to 100 nM protein kinase enzyme in 1× assay buffer
  • Fluorescence‐grade cuvette: e.g., a three‐window fluorescence‐grade quartz cuvette with a path length of 10 mm and aqueous volume of 100 µl (Hellma)
  • Fluorimeter: any standard fluorimeter fitted with temperature control and a normal set of excitation and emission filters will be adequate (e.g., Fluorolog model FL3‐11 fluorimeter; Jobin Yvon, http://www.horiba.com/)

Alternate Protocol 1: Measuring Protein Kinase Activity Using a Fluorescence Plate Reader

  • 96‐well plate (e.g., Corning, catalog # 3992)
  • Multichannel pipettors and solution reservoir (e.g., Eppendorf, catalog no. 022265806)
  • Plate reader: e.g., Victor3 V multi‐label plate reader (Perkin Elmer)
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Figures

Videos

Literature Cited

   Luković, E., González‐Vera, J.A., and Imperiali, B. 2008. Recognition‐domain focused chemosensors: Versatile and efficient reporters of protein kinase activity. J. Am. Chem. Soc. 130:2821‐2827.
   Manning, G., Whyte, D.B., Martinez, R., Hunter, T., and Sudarsanam, S. 2002. The protein kinase complement of the human genome. Science 298:1912‐1934.
   Shults, M.D. and Imperiali, B. 2003. Versatile fluorescent probes of protein kinase activity. J. Am. Chem. Soc. 125:14248‐14249.
   Shults, M.D., Janes, K.A., Lauffenburger, D.A., and Imperiali, B. 2005. A multiplexed homogeneous fluorescence‐based assay for protein kinase activity in cell lysates. Nat. Methods 2:277‐283.
Key Reference
   Jencks, W.P. 1987. Catalysis in Chemistry and Enzymology. Dover Publications, Mineola, New York.
  A good mathematical description of simple kinetic expressions as well as practical considerations.
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