Development of Chemical Probes for Biochemical Detection and Cellular Imaging of Myristoylated and Palmitoylated Proteins

Rami N. Hannoush1

1 Genentech, South San Francisco, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch100143
Online Posting Date:  February, 2011
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Abstract

Fatty acylation of proteins regulates their spatial localization and activity in living cells. Methods to monitor fatty acylation are invaluable for studying its role in regulating protein dynamics. The protocols in this unit describe a procedure that involves metabolic labeling with ω‐alkynyl fatty acids for detecting and cellular imaging of fatty‐acylated proteins, namely myristoylated and palmitoylated proteins. Curr. Protoc. Chem. Biol. 3:15‐26 © 2011 by John Wiley & Sons, Inc.

Keywords: myristoylation; palmitoylation; alkynyl fatty acids; chemical probes; metabolic labeling; click chemistry; fatty acylation

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Biochemical Detection of Myristoylated and Palmitoylated Cellular Proteins by Immunoblotting or In‐Gel Fluorescence
  • Basic Protocol 2: Cellular Imaging of Myristoylated and Plamitoylated Proteins
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Biochemical Detection of Myristoylated and Palmitoylated Cellular Proteins by Immunoblotting or In‐Gel Fluorescence

  Materials
  • Cell line of interest:
    • Examples include:
    • Raw 264.7 macrophages (ATCC #CCL‐2278)
    • MDCK epithelial cells (ATCC #CCL‐34)
    • PC‐3 cells (ATCC #CRL‐1435)
    • Mouse L‐cells (ATCC #CRL‐2648)
    • HeLa cells (ATCC #CCL‐2)
    • Jurkat T cells (ATCC #TIB‐152)
    • COS‐7 cells (ATCC #CRL‐1651)
  • Appropriate cell culture growth medium (e.g., DMEM, F‐12K, RPMI)
  • Fatty acid stock solution (see recipe)
  • Bovine serum albumin (BSA; fatty acid‐free, Sigma‐Aldrich)
  • Dimethyl sulfoxide (DMSO)
  • Ethanol
  • Phosphate‐buffered saline (PBS; see recipe)
  • Lysis buffer (see recipe)
  • BCA protein assay kit (Thermo Scientific)
  • Biotin‐azide or rhodamine‐azide (see recipe)
  • Tris (2‐carboxyethyl)phosphine hydrochloride (TCEP; Sigma‐Aldrich)
  • Tris[(1‐benzyl‐1H‐1,2,3‐triazol‐4‐yl)methyl]amine (TBTA; Sigma‐Aldrich)
  • Copper sulfate (CuSO 4; Sigma‐Aldrich)
  • Acetone, ice‐cold
  • Novex Tris glycine SDS sample buffer (2×) (Invitrogen)
  • NuPAGE sample reducing agent (10×) (Invitrogen)
  • Tris‐glycine gels, precast (Invitrogen)
  • PBS‐T (see recipe)
  • Nonfat dried milk (any grocery store)
  • Streptavidin‐horseradish peroxidase (Invitrogen)
  • Hydroxylamine solution, 50% in water (NH 2OH; Sigma‐Aldrich)
  • Restore western blot stripping buffer (Thermo Scientific)
  • Anti‐β‐tubulin HRP antibody (Invitrogen)
  • 37°C, 5% CO 2 humidified incubator
  • 6‐well plates
  • Sonicator (Branson)
  • Cell scraper (25 cm, Starstedt)
  • Centrifuge (Eppendorf)
  • Nanocep centrifugal ultrafiltration devices (Pall Corporation)
  • Vortexer (VWR)
  • Thermomixer heating block (Eppendorf)
  • ECL immunoblotting detection kit (GE Healthcare)
  • Amersham Hyperfilm ECL (GE Healthcare)
  • Desktop scanner
  • ImageJ or AdobePhotoshop
  • Typhoon scanner
  • Additional reagents and equipment for transferring the proteins onto a nitrocellulose membrane (Gallagher et al., )

Basic Protocol 2: Cellular Imaging of Myristoylated and Plamitoylated Proteins

  Materials
  • Phosphate‐buffered saline (PBS; see recipe)
  • Methanol (Sigma‐Aldrich), prechilled
  • PBS/0.1% (v/v) Triton X‐100
  • Rhodamine‐azide (see recipe)
  • Tris (2‐carboxyethyl)phosphine hydrochloride (TCEP; see recipe)
  • Copper sulfate (CuSO 4; see recipe)
  • Hoechst 33342 (Invitrogen, cat. no. H21492)
  • 12‐well tissue culture plates
  • Glass coverslips, washed in ethanol and left to dry for 10 min under UV light in a tissue culture hood before use
  • 37°C, 5% CO 2 incubator
  • Microscope slides
  • Fluorescence microscope and a 40× or 63× objective
  • Standard image analysis software: e.g., Slidebook 5.0 (Intelligent Imaging Innovation) or ImageJ (NIH)
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Figures

Videos

Literature Cited

Literature Cited
   Drisdel, R.C. and Green, W.N. 2004. Labeling and quantifying sites of protein palmitoylation. Biotechniques 36:276‐285.
   Gallagher, S., Winston, S.E., Fuller, S.A., and Hurrell, J.G. 2008. Immunoblotting and Immunodetection. Curr. Protoc. Mol. Biol. 83:10.8.1‐10.8.28.
   Hannoush, R.N. and Arenas‐Ramirez, N. 2009. Imaging the lipidome: Omega‐alkynyl fatty acids for detection and cellular visualization of lipid‐modified proteins. ACS Chem. Biol. 4:581‐587.
   Hannoush, R.N. and Sun, J. 2010. The chemical toolbox for monitoring protein fatty acylation and prenylation. Nat. Chem. Biol. 6:498‐506.
   Heal, W.P., Wickramasinghe, S.R., Bowyer, P.W., Holder, A.A., Smith, D.F., Leatherbarrow, R.J., and Tate, E.W. 2008. Site‐specific N‐terminal labelling of proteins in vitro and in vivo using N‐myristoyl transferase and bioorthogonal ligation chemistry. Chem. Commun. (Camb) 4:480‐482.
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   Lewis, W.G., Magallon, F.G., Fokin, V.V., and Finn, M.G. 2004. Discovery and characterization of catalysts for azide−alkyne cycloaddition by fluorescence quenching. J. Am. Chem. Soc. 126:9152‐9153.
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   Peseckis, S.M., Deichaite, I., and Resh, M.D. 1993. Iodinated fatty acids as probes for myristate processing and function. Incorporation into pp60v‐src. J. Biol. Chem. 268:5107‐5114.
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   Schlesinger, M.J., Magee, A.I., and Schmidt, M.F.G. 1980. Fatty acid acylation of proteins in cultured cells. J. Biol. Chem. 255:10021‐10024.
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   Wang, Q., Chan, T.R., Hilgraf, R., Fokin, V.V., Sharpless, K.B., and Finn, M.G. 2003. Bioconjugation by copper(I)‐catalyzed azide‐alkyne [3 + 2] cycloaddition. J. Am. Chem. Soc. 125:3192‐3193.
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