Metabolic Labeling with Amino Acids

互联网2013-12-31

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Abstract
Table of Contents
Materials
Literature Cited

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 [³⁵ S]?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.

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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

[³⁵ S]L‐Methionine (>800 Ci/mmol) or [³⁵ S]‐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% CO₂ 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/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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Figures

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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.