Metabolic Labeling with Amino Acids

Juan S. Bonifacino1

1 National Institute of Child Health and Human Development, Bethesda, Maryland
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 7.1
DOI:  10.1002/0471143030.cb0701s00
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Metabolic labeling techniques are used to study the biosynthesis, processing, intracellular transport, secretion, degradation, and physical‐chemical properties of proteins. This unit provides protocols for metabolic labeling of cells with [35S]‐labeled amino acids to provide material suitable for analysis by immunoprecipitation, for characterization of cellular proteins, for analysis of protein trafficking, and for one‐ and two‐dimensional gel electrophoresis. Three procedures are described for suspension and adherent cells: pulse‐labeling, pulse‐chase labeling, or continuous long‐term labeling. A support protocol describes TCA precipitation to measure the extent of labeling.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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, T and B cell hybridomas), grown in a humidified, 37°C, 5% CO 2 incubator or prepared from tissues (e.g., lymphocytes, unit 2.2)
  • PBS ( appendix 2A), 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; units 2.1 & 2.3)
  • 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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

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.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library