Analysis of Protein Acylation

Ruth Zeidman1, Caroline S. Jackson1, Anthony I. Magee1

1 Molecular Medicine, National Heart & Lung Institute, Imperial College London, London
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.2
DOI:  10.1002/0471140864.ps1402s55
Online Posting Date:  February, 2009
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Proteins can be acylated with a variety of fatty acids attached by different covalent bonds, influencing, among other things, their function and intracellular localization. This unit describes methods to analyze protein acylation, both levels of acylation and also the identification of the fatty acid and the type of bond present in the protein of interest. Protocols are provided for metabolic labeling of proteins with tritiated fatty acids, for exploitation of the differential sensitivity to cleavage of different types of bonds, in order to distinguish between them, and for thin‐layer chromatography to separate and identify the fatty acids associated with proteins. Curr. Protoc. Protein Sci. 55:14.2.1‐14.2.12. © 2009 by John Wiley & Sons, Inc.

Keywords: acylation; palmitoylation; myristoylation; fatty acids; protein modification

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Table of Contents

  • Introduction
  • Basic Protocol 1: Biosynthetic Labeling with Fatty Acids
  • Basic Protocol 2: Analysis of Fatty Acid Linkage to Protein
  • Basic Protocol 3: Analysis of Total Protein‐Bound Fatty Acid Label in Cell Extract
  • Basic Protocol 4: Analysis of Fatty Acid Label Identity
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Biosynthetic Labeling with Fatty Acids

  • Cells for culture (also see appendix 3C)
  • Complete tissue culture medium appropriate for cells (also see appendix 3C)
  • Labeling medium: complete tissue culture medium containing the appropriate dialyzed serum and 1 mM sodium pyruvate, 37°C
  • 5 to 10 µCi/µl [9,10(n)‐3H]fatty acid, e.g., [9,10(n)‐3H]palmitic acid or [9,10(n)‐3H]myristic acid (30 to 60 Ci/mmol; Amersham GE Healthcare, American Radiolabeled Chemicals, or NEN PerkinElmer) in ethanol
  • Phosphate‐buffered saline (PBS), pH 7.2 ( appendix 2E), ice‐cold
  • Nitrogen gas
  • 1% (w/v) SDS or SDS sample buffer (for SDS‐PAGE, when using adherent or nonadherent cells respectively; unit 10.1) or RIPA lysis buffer (for immunoprecipitation; unit 13.2)
  • 5× SDS sample buffer (see recipe)
  • Cell scrapers
  • Additional reagents and equipment for cell culture ( appendix 3C), immunoprecipitation (unit 13.2), SDS‐PAGE (unit 10.1), treating a gel with sodium salicylate (unit 14.3) or DMSO/PPO solution (unit 10.2), and fluorography (unit 10.2)
NOTE: All reagents and equipment coming into contact with live cells must be sterile, and proper sterile technique should be used accordingly.NOTE: All culture incubations are performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified.

Basic Protocol 2: Analysis of Fatty Acid Linkage to Protein

  • Lysate or immunoprecipitate from [3H]fatty acid‐labeled cells (see protocol 1, step 6)
  • 0.2 M potassium hydroxide (KOH) in methanol
  • Methanol
  • 1 M hydroxylamine⋅HCl, titrated to pH 7.5 with NaOH
  • 1 M Tris⋅Cl, pH 7.5 ( appendix 2E)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1), treating a gel with sodium salicylate (unit 14.3) or DMSO/PPO solution (unit 10.2), and fluorography (unit 10.2)

Basic Protocol 3: Analysis of Total Protein‐Bound Fatty Acid Label in Cell Extract

  • 0.1 M HCl/acetone, −20°C
  • Lysate from [3H]fatty acid‐labeled cells in 1% SDS (see protocol 1, step 4a or 5b)
  • 1% (w/v) SDS
  • 2:1 (v/v) chloroform/methanol
  • Diethyl ether
  • Nitrogen gas
  • 6 M HCl (concentrated HCl diluted 1:1 with H 2O)
  • Hexane
  • 5 to 10 µCi/µl [9,10(n)‐3H]fatty acid standards (30 to 60 Ci/mmol; Amersham GE Healthcare, American Radiolabeled Chemicals, or NEN PerkinElmer) in ethanol
  • 90:10 (v/v) acetonitrile/acetic acid
  • EN3HANCE spray (PerkinElmer)
  • 15‐ml polypropylene centrifuge tubes
  • Mistral 3000i benchtop centrifuge with swing‐out four‐bucket rotor or equivalent
  • 30‐ml thick‐walled Teflon container with an air‐tight screw top
  • 110°C oven
  • Thin‐layer chromatography tank
  • RP18 thin‐layer chromatography plate (e.g., Merck)
  • Kodak BioMax MS film, preflashed

Basic Protocol 4: Analysis of Fatty Acid Label Identity

  • SDS‐PAGE gel of lysate from [3H]fatty acid‐labeled cells ( protocol 1)
  • Additional reagents and equipment for analysis of protein‐bound label (see protocol 3)
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Key Reference
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  A compilation of methods used in studying lipid modification of proteins.
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