In‐Gel Phosphatase Assay Using Fluorogenic and Radioactive Substrates

Isamu Kameshita1

1 Kagawa University, Kagawa, Japan
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 13.12
DOI:  10.1002/0471140864.ps1312s65
Online Posting Date:  August, 2011
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Abstract

To investigate the regulatory mechanisms of cellular signaling by protein phosphorylation, it is important to analyze protein phosphatases, as well as protein kinases expressed in cells and tissues. In this unit, two different types of in‐gel phosphatase assays are described. The first is an in‐gel phosphatase assay using fluorogenic substrates. Protein samples containing phosphatase activities are resolved by native polyacrylamide gel electrophoresis (native‐PAGE) and phosphatase activities detected in situ using fluorogenic substrates, such as 4‐methylumbelliferyl phosphate (MUP) or 6,8‐difluoro‐4‐methylumbelliferyl phosphate (DiFMUP). The other assay is an in‐gel phosphatase assay using 32P‐labeled substrates. In this method, protein samples are resolved by SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) using polyacrylamide gels containing 32P ‐labeled substrates, renatured in situ, and the dephosphorylating activities detected by autoradiography. Each method has advantages and disadvantages that are discussed in the commentary. Curr. Protoc. Protein Sci. 65:13.12.1‐13.12.10. © 2011 by John Wiley & Sons, Inc.

Keywords: in‐gel assay; phosphatase; polyacrylamide gel electrophoresis

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In‐Gel Phosphatase Assay Using Fluorogenic Substrates
  • Basic Protocol 2: In‐Gel Phosphatase Assay Using Radioactive Substrates
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: In‐Gel Phosphatase Assay Using Fluorogenic Substrates

  Materials
  • Protein sample: Tissue extracts or cell lysates (e.g., rat tissue extracts)
  • Homogenization buffer (see recipe)
  • 6× sample buffer for native‐PAGE (see recipe)
  • Alkaline phosphatase from calf intestine (CIP; e.g., Roche)
  • Reaction mixture for fluorescent assay (see recipe)
  • Kimwipes
  • Heat‐sealable plastic bag (e.g., Hybri‐bag from CosmoBio)
  • Disposable gloves
  • 30°C incubator
  • Transilluminator (excitation wavelength at 365 nm)
  • Additional reagents and equipment for native‐PAGE (unit 10.3)

Basic Protocol 2: In‐Gel Phosphatase Assay Using Radioactive Substrates

  Materials
  • Poly‐L‐lysine [Poly(Lys); 40 to 100 kDa; see recipe]
  • Sodium phosphate buffer, pH 7.2 ( appendix 2E)
  • N‐(6‐meleimidocaproyloxy)succinimide (Dojindo Laboratories)
  • Dimethyl formamide
  • Dichloromethane
  • CaMKII(281‐289): Met‐His‐Arg‐Gln‐Glu‐Thr‐Val‐Asp‐Cys [We prepared this peptide using a Shimadzu PSSM‐8 automated peptide synthesizer and purified it by reverse‐phase HPLC; otherwise, we can obtain high‐quality peptides from various providers of custom peptides (e.g., Invitrogen, CosmoBio, Promega, and Peptide Institute)]
  • Peptide solution (see recipe)
  • Dithiothreitol (DTT)
  • HEPES‐NaOH buffer, pH 8.0 (see recipe)
  • EGTA
  • Magnesium acetate
  • [γ‐32P]ATP
  • 30K‐CaMKII: constitutively active form of Ca2+/calmodulin‐dependent protein kinase II (CaMKII) (Shoju et al., )
  • Myelin basic protein (Sigma)
  • Catalytic subunit of cAMP‐dependent protein kinase (Sigma)
  • 10% acrylamide solution
  • Enzyme samples containing phosphatase activity (e.g., rat tissue extracts)
  • SDS sample buffer
  • 20% 2‐propanol in 50 mM Tris⋅Cl, pH 7.0
  • 50 mM Tris⋅Cl, pH 7.0 ( appendix 2E)
  • 2‐mercaptoethanol (2‐ME)
  • Denaturation buffer (see recipe)
  • Renaturation buffer (see recipe)
  • Coomassie blue solution (see recipe)
  • 7% acetic acid
  • 2‐ml round‐bottom microcentrifuge tubes (e.g., Eppendorf microtubes)
  • Small stirring bar
  • Vortex
  • Microcentrifuge
  • Rocking platform shaker
  • X‐ray film
  • Film cassette
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1) and autoradiography (unit 10.11)
NOTE: For preparation of a peptide conjugate, a peptide with a cysteinyl residue at the amino‐ or carboxyl‐terminal should be used.
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Figures

Videos

Literature Cited

Literature Cited
   Bischoff, K.M., Shi, L., and Kennelly, P.J. 1998. The detection of enzyme activity following sodium dodecyl sulfate polyacrylamide gel electrophoresis. Anal. Biochem. 260:1‐17.
   Burridge, K. and Nelson, A. 1995. An in‐gel assay for protein tyrosine phosphatase activity: Detection of widespread distribution in cells and tissues. Anal. Biochem. 232:56‐64.
   Ishida, A., Kameshita, I., and Fujisawa, H. 1998. A novel protein phosphatase that dephosphorylates and regulates Ca2+/calmodulin‐dependent protein kinase II. J. Biol. Chem. 273:1904‐1910.
   Kameshita, I. and Fujisawa, H. 1989. A sensitive method for detection of calmodulin‐dependent protein kinase II activity in sodium dodecylsulfate polyacrylamide gel. Anal. Biochem. 183:139‐143.
   Kameshita, I. and Fujisawa, H. 1996. Detection of protein kinase activities toward oligopeptides in sodium dodecyl sulfate‐polyacrylamide gel. Anal. Biochem. 237:198‐203.
   Kameshita, I., Ishida, A., Okuno, S., and Fujisawa, H. 1997. Detection of protein phosphatase activities in sodium dodecyl sulfate‐polyacrylamide gel using peptide substrates. Anal. Biochem. 245:149‐153.
   Kameshita, I., Baba, H., Umeda, Y., and Sueyoshi, N. 2010. In‐gel phosphatase assay using fluorogenic substrates. Anal. Biochem. 400:118‐122.
   Kinoshita, S., Sueyoshi, N., Shoju, H., Suetake, I., Nakamura, M., Tajima, S., and Kameshita, I. 2004. Cloning and characterization of a novel Ca2+/calmodulin‐dependent protein kinase I homologue in Xenopus laevis. J. Biochem. 135:619‐630.
   Shoju, H., Sueyoshi, N., Ishida, A., and Kameshita, I. 2005. High level expression and preparation of autonomous Ca2+/calmodulin‐dependent protein kinase II in Escherichia coli. J. Biochem. 138:605‐611.
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