Metabolic Labeling with Amino Acids

Juan S. Bonifacino1

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

  • Safety Precautions for Working With 35s‐Labeled Compounds
  • Basic Protocol 1: 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 1: TCA Precipitation to Determine Label Incorporation
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: 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% CO 2 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 protocol 6)

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

  • 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

  • Chase medium (see recipe), 37°C

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

  • Long‐term labeling medium (see recipe), warmed to 37°C
  • 75‐cm2 tissue culture flask

Alternate Protocol 4: Metabolic Labeling with Other Radiolabeled Amino Acids

  Materials
  • Labeled cell suspension (see protocol 1 or Alternate Protocols protocol 21 to protocol 54)
  • BSA/NaN 3: 1 mg/ml BSA containing 0.02% (w/v) sodium azide (NaN 3)
  • 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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Figures

Videos

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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