Biosynthetic Labeling of Proteins

Juan S. Bonifacino1

1 National Institute of Child Health and Human Development, Bethesda, Maryland
Publication Name:  Current Protocols in Immunology
Unit Number:  Unit 8.12
DOI:  10.1002/0471142735.im0812s41
Online Posting Date:  May, 2001
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Abstract

Biosynthetic labeling techniques are commonly used in the study of biochemical properties, synthesis, processing, intracellular transport, secretion, and degradation of proteins. In this unit, protocols are described for biosynthetically labeling many secreted and membrane proteins. The describes short‐term labeling of cells in suspension (30 min to 3 hr) and an alternate protocol describes a modification of this procedure to be used for adherent cells. Alternate protocols for long‐term (3 to 24 hr) and pulse‐chase labeling of cells are also presented. Label incorporation can be determined as described in a support protocol. The cells used in these protocols can be either from suspension cultures or from single‐cell suspensions of spleen or thymus.

     
 
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Table of Contents

  • Basic Protocol 1: Short‐Term Labeling of Cells in Suspension with [35S ]Methionine
  • Alternate Protocol 1: Short‐Term Labeling of Adherent Cells with [35 S]Methionine
  • Alternate Protocol 2: Long‐Term Labeling of Cells with [35S]Methionine
  • Alternate Protocol 3: Pulse‐Chase Labeling of Cells with [35S]Methionine
  • Alternate Protocol 4: Biosynthetic Labeling with Other Amino Acids
  • Support Protocol 1: TCA Precipitation to Determine Label Incorporation
  • Reagents and Solutions
  • Commentary
  • Tables
     
 
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Materials

Basic Protocol 1: Short‐Term Labeling of Cells in Suspension with [35S ]Methionine

  Materials
  • recipeShort‐term labeling medium, warmed to 37°C in a water bath
  • Cells in suspension grown in a humidified 37°C, 5% CO 2 incubator
  • [35S]L‐methionine (800 to 1200 Ci/mmol; see critical parameters for caution)
  • Phosphate‐buffered saline (PBS; appendix 2A), ice‐cold
  • 15‐ or 50‐ml conical tubes with screwcaps, sterile
CAUTION: Volatile 35S‐containing compounds can be released during the labeling procedure (refer to Safety Precautions for Radioisotopes in appendix 1Q).

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

  Additional Materials
  • Adherent cells, grown in a humidified 37°C, 5% CO 2 incubator
  • 100‐mm‐diameter tissue culture dishes
  • Disposable plastic scraper or rubber policeman
  • 15‐ml conical tubes

Alternate Protocol 2: Long‐Term Labeling of Cells with [35S]Methionine

  Additional Materials
  • recipeLong‐term labeling medium, warmed to 37°C in a water bath
  • 150‐cm2 flask

Alternate Protocol 3: Pulse‐Chase Labeling of Cells with [35S]Methionine

  Additional Materials
  • recipeChase medium, 37°C

Alternate Protocol 4: Biosynthetic Labeling with Other Amino Acids

  Additional Materials
  • 0.1 mg/ml bovine serum albumin (BSA) containing 0.02% NaN 3
  • recipe20% and 10% TCA, ice‐cold
  • 100% ethanol
  • Filtration apparatus with vacuum line
  • Glass microfiber filters, 2.5‐cm diameter (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

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Literature Cited

Literature Cited
   Coligan, J.E., Gates, III F.T., Kimball, E.S., and Maloy, W.L. 1983. Radiochemical sequence analysis of biosynthetically labeled proteins. Methods Enzymol. 91:413‐434.
   Cuello, A.C., Priestly, J.V., and Milstein, C. 1982. Immunocytochemistry with internally labeled monoclonal antibodies. Proc. Natl. Acad. Sci. U.S.A. 79:665‐669.
   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 (Lond.) 335:120.
   Robb, R.J., Munck, A., and Smith, K.A. 1981. T cell growth factor receptors. Quantitation, specificity, and biological relevance. J. Exp. Med. 154:1455‐1474.
Key Reference
   Coligan et al., 1983. See above.
  Contains a detailed description of conditions used to biosynthetically label proteins with different radioactive amino acids.
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