Assays for Acetylation and Other Acylations of Lysine Residues

Nadine Pelletier1, Serge Grégoire1, Xiang‐Jiao Yang1

1 Goodman Cancer Research Center and Department of Medicine, McGill University, Montreal, Quebec
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 14.11
DOI:  10.1002/cpps.26
Online Posting Date:  February, 2017
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Abstract

Lysine acetylation refers to addition of an acetyl moiety to the epsilon‐amino group of a lysine residue and is important for regulating protein functions in various organisms from bacteria to humans. This is a reversible and precisely controlled covalent modification that either serves as an on/off switch or participates in a codified manner with other post‐translational modifications to regulate different cellular and developmental processes in normal and pathological states. This unit describes methods for in vitro and in vivo determination of lysine acetylation. Such methods can be easily extended for analysis of other acylations (such as propionylation, butyrylation, crotonylation, and succinylation) that are also present in histones and many other proteins. © 2017 by John Wiley & Sons, Inc.

Keywords: butyrylation; chromatin; crotonylation; glutarylation; histone acetyltransferase; lysine acetylation; metabolic enzymes; propionylation; transcription factor; tubulin

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: In Vitro Protein Acetylation Determination by P81 Filter Retention
  • Basic Protocol 2: In Vitro Protein Acetylation Analysis by Autoradiography
  • Alternate Protocol 1: In Vitro Protein Acetylation Analysis by Fluorography
  • Basic Protocol 3: Detection of In Vivo Protein Acetylation by Anti–Acetyl Lys Antibodies
  • Alternate Protocol 2: Detection of In Vivo Protein Acetylation by [3H]Acetate Labeling
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: In Vitro Protein Acetylation Determination by P81 Filter Retention

  Materials
  • 5× buffer A (see recipe and Commentary for discussion on the buffer composition)
  • 2 mg/ml mixed histones (calf thymus, type IIa; Sigma) or core histone octamers, mononucleosomes, and oligonucleosomes (see annotation to step 4) in 1 mM HCl
  • 250 μCi/ml [3H]acetyl CoA (2 to 10 Ci/mmol, Amersham Biosciences, cat. no. TRK688)
  • Lysine acetyltransferase (25 to 200 ng; see discussion in protocol introduction above)
  • P81 filter washing buffer (see recipe)
  • 1:2 (v/v) methanol/chloroform (optional)
  • Acetone (optional)
  • Biodegradable counting scintillant (Amersham Bioscience, or equivalents)
  • P81 phosphocellulose filter paper (Whatman, cat. no. 3698‐865 or ‐915)
  • Regular chromatography filter paper (Fisher Scientific)
  • 30°C thermostatted water bath
  • 37ºC reciprocal shaking water bath (Precision Scientific, or equivalent)
  • 0.5‐ml microcentrifuge tubes, sterile
  • 2‐liter jar
  • Scintillation vials (Fisher Scientific, or equivalent)
  • Liquid scintillation counter

Basic Protocol 2: In Vitro Protein Acetylation Analysis by Autoradiography

  Materials
  • 5× Buffer A (see recipe)
  • 2 mg/ml mixed histones (calf thymus, type IIa; Sigma) or core histones and nucleosomes, purified from HeLa S3 cells (see protocol 1) or core histones and nucleosomes purified from HeLa S3 cells (see annotation to protocol 1, step 4) in 1 mM HCl
  • 50 μCi/ml [1‐14C]acetyl CoA (55 mCi/mmol; Amersham Biosciences, cat. no. CFA729)
  • Lysine acetyltransferase (25 to 200 ng; also see protocol 1)
  • 3× SDS sample buffer (see recipe)
  • Pre‐stained protein molecular weight standards
  • Coomassie brilliant blue R‐250 staining solution (see recipe)
  • Destaining solution (see recipe)
  • 0.5‐ml microcentrifuge tubes, sterile
  • 30°C thermostatted water bath
  • 1‐liter beaker of boiling water (∼300 ml)
  • Clear plastic wrap (e.g., Saran wrap)
  • Gel dryer (e.g., BioRad, model 583)
  • Diaphragm‐type vacuum pump (Vacuubrand GMBH+ Co., type MZ 2 C)
  • Chromatography paper
  • Phosphor imager: FUJIX BAS 100 (Fuji) or Typhoon 8600 (Molecular Dynamics)
  • Radioactive ink (prepared by arbitrarily mixing some radioactive material into a drop of ink)
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher, )

Alternate Protocol 1: In Vitro Protein Acetylation Analysis by Fluorography

  Additional Materials (also see protocol 2)
  • Amplify fluorographic reagent (GE Healthcare Life Sciences, cat no. NAMP 100) or Enlighting solution (NEN Life Science, cat no. NEF‐974)

Basic Protocol 3: Detection of In Vivo Protein Acetylation by Anti–Acetyl Lys Antibodies

  Materials
  • Human embryonic kidney (HEK) 293 cells (ATCC #CRL‐1573)
  • Complete DMEM medium ( appendix 3C; Phelan, )
  • Transfection reagent (Superfect from Qiagen, or equivalent)
  • Complete DMEM ( appendix 3C; Phelan, ) containing 3 μM trichostatin A (TSA; Sigma) and 5 mM nicotinamide (Sigma)
  • Phosphate‐buffered saline (PBS; see recipe), cold
  • PBS (see recipe) containing 1 μM trichostatin A (TSA; Sigma) and 5 mM nicotinamide (Sigma)
  • Buffer K (see recipe)
  • Buffer K (see recipe) containing 1 μM trichostatin A (TSA; Sigma) and 5 mM nicotinamide
  • M2 agarose beads (Sigma)
  • Buffer K (see recipe) containinng 1 μM trichostatin A (TSA; Sigma), 5 mM nicotinamide, and 2.5 μl of 2 mg/ml FLAG peptide (prepare 2 mg/ml FLAG peptide in 0.1 M Tris⋅Cl, pH 8.0)
  • 3× SDS sample buffer (see recipe)
  • Blocking solution (see recipe)
  • PBST (see recipe)
  • Primary antibodies:
    • Anti‐FLAG (rabbit) antibody (Sigma)
    • Anti‐acetyl Lys (rabbit) antibody against specific residues of target protein (commercially available Abcam and EMD Millipore, or developed in‐house using acetyl Lys peptides)
    • Anti‐acetyl Lys rabbit polyclonal antibody (e.g., Cell Signaling Technology and ImmuneChem Pharmaceuticals Inc.)
    • Secondary antibody: anti‐rabbit IgG, horseradish peroxidase‐linked whole antibody (Amersham Biosciences)
  • Chemiluminescent substrate (Supersignal from Pierce, or equivalent)
  • 10‐cm culture plates
  • Probe sonicator (e.g., Virsonic from VirTis)
  • End‐over‐end rotator
  • 0.5 ml tubes, pre‐chilled
  • Boiling water bath
  • Nitrocellulose blotting membrane (Pall Life Sciences, BioTrace NT)
  • Platform rocker
  • Additional reagents and equipment for SDS‐PAGE (unit 10.1; Gallagher, ), blot transfer (unit 10.7; Goldman et al., ), and immunoblot detection (unit 10.10; Gallagher, )

Alternate Protocol 2: Detection of In Vivo Protein Acetylation by [3H]Acetate Labeling

  Additional Materials (also see protocol 4 and protocol 3)
  • 10 mCi/ml [3 H]acetate, sodium salt (2 to 5 Ci/mmol; Perkin Elmer)
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Figures

Videos

Literature Cited

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