35S Methionine Labeling

Overview

Metabolic labeling of cells with low-energy beta-emitting radioisotopes such as 35S-methionine and 35S-methionine/cysteine is often used to follow the biosynthesis, maturation, and degradation of proteins in vivo. Labeling of methionine-containing proteins in cells relies on the use of methionine-free culture media supplemented with a radiolabeled source of 35S. Quantification and identification of the labeled proteins are usually carried out by separation on two-dimensional SDS-PAGE gel and exposing to film (autoradiography) or phosphorimaging (though other methods of detection are possible).

Cell-free protein translation using reticulocyte lysates or wheat germ extracts is also performed using 35S-methionine. Cell-free translation provides a quick way to make small amounts of labeled protein quickly, for use in various types of biochemical assays.

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What do I need to run this assay?

Cell labeling assay

  • 35S-labeled methionine (refer to table in next section)
  • Cells
  • Methionine-free culture media
  • PBS
  • DNase/RNase solution
  • Tissue culture equipment
  • Gel electrophoresis reagents and equipment
  • Film for autoradiography - see Products and Catalog numbers below (unless using a phosphorimager)
  • Film developer, or a phosphorimager (such as Cyclone™ phosphorimager) as appropriate
  • Recommended: Charcoal trap (Cat. No. NEX033T)


Cell-free translation

  • 35S-labeled methionine (refer to table in next section)
  • Cell-free translation system (typically using a reticulocyte lysate or wheat germ extract, such as the TNT® systems from Promega)
  • Template RNA (if using an in vitro translation system) or DNA (if using a coupled in vitro transcription/translation system)
  • Ribonuclease inhibitor (such as RNasin)
  • Recommended: Charcoal trap (Cat. No. NEX033T)

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Products and catalog numbers

methionine_chart_ASK.jpg
Methionine selection chart


Radiolabeled 35S Methionine and 35S Methionine/Cysteine products
Refer to the flow chart above for guidance in choosing a product for your assay. You can also visit Which formulation should I choose? for additional help to select the appropriate product.


Precautions for handling 35S-labeled compounds

  • Solutions containing 35S labeled compounds can release volatile radioactive by-products. In addition to the usual safety practices for handling radioactive material, it is highly recommended to open the vial with a charcoal trap (Cat. No. NEX033T)
  • It is advisable when incubating cells with 35S methionine and cysteine that you have charcoal present to capture the volatile by-products. Activated charcoal is available from a variety of vendors.


Autoradiography enhancers for film or phosphorimaging

  • EN3HANCE® Autoradiography Enhancer (Cat. No. 6NE9701): use on gels for best band resolution
  • ENLIGHTNING™ Rapid Autoradiography Enhancer (Cat. No. 6NE9741): safer alternative to 6NE9701. Use on gels.

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Protocol-in-brief

Cell labeling

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Protocol

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Citations

Cell labeling

  1. Bizios, N. Eukaryotic cell labeling and preparation for 2-D. Methods Mol. Biol112, 49-52 (1999). Link
  2. Gonnord, P. et al. Palmitoylation of the P2X7 receptor, an ATP-gated channel, controls its expression and association with lipid rafts. FASEB J23, 795-805 (2009). Link
  3. Kolli, B.K., Kostal, J., Zaborina, O., Chakrabarty, A.M. & Chang, K. Leishmania-released nucleoside diphosphate kinase prevents ATP-mediated cytolysis of macrophages. Mol. Biochem. Parasitol158, 163-175 (2008). Link
  4. Pollard, J.W. The in vivo isotopic labeling of proteins for polyacrylamide gel electrophoresis. Methods Mol. Biol32, 67-72 (1994). Link
  5. Takahashi, M. & Ono, Y. Pulse-chase analysis of protein kinase C. Methods Mol. Biol233, 163-170 (2003). Link


Cell-free translation

  1. Kraichely, D.M. & MacDonald, P.N. Confirming yeast two-hybrid protein interactions using in vitro glutathione-S-transferase pulldowns. Methods Mol. Biol177, 135-150 (2001). Link
  2. Movahedzadeh, F., Rico, S.G. & Cox, R.A. In vitro transcription and translation. Methods Mol. Biol235, 247-255 (2003). Link
  3. Wilson, C.M. & Bulleid, N.J. Investigation of folding and degradation of mutant proteins synthesized in semipermeabilized cells. Methods Mol. Biol232, 295-312 (2003). Link

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