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Metabolic Labeling with Amino Acids

Juan S. Bonifacino1

1National Institute of Child Health and Human Development, NIH, Bethesda, Maryland

Publication Name: 
Current Protocols in Protein Science
Unit Number: 
Unit 3.7
DOI: 
10.1002/0471140864.ps0307s17
Online Posting Date: 
May, 2001
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Abstract

Metabolic labeling techniques are used to study biosynthesis, processing, intracellular transport, secretion, degradation, and physical-chemical properties of proteins. This unit focuses on pulse-labeling and pulse-chase experiments done with [35S]-methionine, but gives directions for labeling with other radiolabeled amino acids, and also offers guidance for the safe use and handling of 35S-labeled compounds.

     
 
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Table of Contents

  • Unit Introduction
  • Safety Precautions for Working With 35s-Labeled Compounds
  • Basic Protocol: Pulse-Labeling of Cells in Suspension with [35S]Methionine
  • Alternate Protocol 1: Pulse-Labeling of Adherent Cells with [35S]Methionine
  • Alternate Protocol 2: Pulse-Chase Labeling of Cells with [35S]Methionine
  • Alternate Protocol 3: Long-Term Labeling of Cells with [35S]Methionine
  • Alternate Protocol 4: Metabolic Labeling with Other Radiolabeled Amino Acids
  • Support Protocol: TCA Precipitation to Determine Label Incorporation
  • Reagents and Solutions
  • Commentary
  • Bibliography
  • Tables
     
 
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Materials

Basic Protocol: Pulse-Labeling of Cells in Suspension with [35S]Methionine

 Materials
  • [35S]l-Methionine (>800 Ci/mmol) or 35S-labeled protein hydrolyzate (>1000 Ci/mmol)
  • Pulse-labeling medium (see recipe), warmed to 37°C
  • Cell suspension (e.g., Jurkat, RBL, K562, BW5147, or T or B cell hybridomas), grown in a humidified 37°C, 5% CO2 incubator or prepared from tissues (e.g., lymphocytes)
  • PBS (appendix 2E), ice cold
  • Vacuum aspirator with trap for liquid radioactive waste
  • Additional reagents and equipment for TCA precipitation (optional; see Support Protocol)

Alternate Protocol 1: Pulse-Labeling of Adherent Cells with [35S]Methionine

 Additional Materials (also see Basic Protocol)
  • Adherent cells (e.g., HeLa, NRK, M1, COS-1, CV-1, or fibroblasts or endothelial cells in primary culture)
  • 100-mm tissue culture dishes

Alternate Protocol 2: Pulse-Chase Labeling of Cells with [35S]Methionine

 Additional Materials (also see Basic Protocol and Alternate Protocol 1)
  • Chase medium (see recipe), 37°C

Alternate Protocol 3: Long-Term Labeling of Cells with [35S]Methionine

 Additional Materials (also see Basic Protocol and Alternate Protocol 1)
  • Long-term labeling medium (see recipe), warmed to 37°C
  • 75-cm2 tissue culture flask

Support Protocol: TCA Precipitation to Determine Label Incorporation

 Materials
  • Labeled cell suspension (see Basic Protocol or Alternate Protocols 1 to 4)
  • BSA/NaN3: 1 mg/ml BSA containing 0.02% (w/v) sodium azide (NaN3)
  • 10% (w/v) TCA solution (see recipe), ice cold
  • Ethanol
  • Filtration apparatus attached to a vacuum line
  • 2.5-cm glass microfiber filter disks (Whatman GF/C)
CAUTION: TCA is extremely caustic. Protect eyes and avoid contact with skin when preparing and handling TCA solutions.
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Literature Cited

 Literature Cited
    Braakman, I., Hoover-Litty, H., Wagner, K.R., and Helenius, A. 1991. Folding of influenza hemagglutinin in the endoplasmic reticulum. J. Cell Biol. 114:401-411.
    Coligan, J.E., Gates, F.T. III., Kimball, E.S., and Maloy, W.L. 1983. Radiochemical sequence analysis of metabolically labeled proteins. Methods Enzymol. 91:413-434.
    Lathe, R. 1985. Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations. J. Mol.Biol. 183:1-12.
    Meisenhelder, J. and Hunter, T. 1988. Radioactive protein labelling techniques. Nature 335:120.
 Key Reference
    Coligan et al., 1983. See above.

Contains a detailed description of conditions used to metabolically label proteins with different radiolabeled amino acids.

     
 
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