Huh-7 cells were passaged eight times in light or heavy label media, i.e., Arg- and Lys-free DMEM (PAA Laboratories) supplemented with 2 mM L-glutamine, 1 mM pyruvate, 10% dialyzed FBS, 0.375% sodium bicarbonate, 48 μg/ml Arg (Arg-0, Arg-6, and Arg-10, respectively), and 73 μg/ml Lys (Lys-0 and Lys-8, respectively; Cambridge Isotope Labs). Confluent P150 cultures were incubated with J6/JFH clone 2 (MOI 10 after 1:5 dilution in serum-free label media) for 15 min at 37°C or treated in a similar manner with virus-free conditioned cell culture media processed in the same way as the HCV preparation (mock electroporation). One-step immunoprecipitations of membrane proteins were performed as detailed in the Supplemental Experimental Procedures . Experiments were conducted in four replicates (two heavy- and two light-labeled cultures per experimental condition).
Arg 6
Manufactured by Cambridge Isotopes
Arg-6 is a stable isotope-labeled amino acid that contains six atoms of the isotope carbon-13 in the arginine molecule. This product is intended for use in research and analytical applications that require the use of stable isotope-labeled compounds.
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Lab products found in correlation
2 protocols using arg 6
1
Stable Isotope Labeling of Huh-7 Cells for HCV Immunoprecipitation
2
SILAC-Based U2OS Cell Line Generation
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U2OS and U2OS Flp-In T-Rex cells were grown as adherent cells in Dulbecco's modified eagle medium (DMEM) depleted of arginine and lysine (Life Technologies, Carlsbad, CA A14431–01) supplemented with 10% dialyzed fetal bovine serum (Invitrogen, Carlsbad, CA 26400–044), 100 U/ml penicillin/streptomycin and 2 mm GlutaMax. Arginine and lysine were added in either light (Arg0, Sigma, A5006; Sigma-Aldrich (St-Louis, MO) Lys0, Sigma, L5501), medium (Arg6, Cambridge Isotope Laboratories, Inc. (Tewksbury, MA) (CIL), CNM-2265; Lys4, CIL, DLM-2640), or heavy (Arg10, CIL, CNLM-539; Lys8, CIL, CNLM-291) form to a final concentration of 28 μg/ml for arginine and 49 μg/ml for lysine. l -proline was added to a final concentration of 10 μg/ml to prevent arginine to proline conversion (see supplemental Fig. 1). Proteins were tested for >99% incorporation of the label after six passages by mass spectrometry (data not shown). U2OS stable cell lines were generated by transfecting pgLAP1 plasmids containing the cDNA of interest along with pOG44, the plasmid expressing the Flp-recombinase. Cells were then cultured with the addition of 150 μg/ml Hygromycin B and 15 μg/ml Blasticidine-HCl. For induction of DNA damage, the topoisomerase II inhibitor etoposide (#E1383, Sigma-Aldrich) was used at the indicated concentrations for 1 h, followed by wash with PBS and fresh normal media added.
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