Silac

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SILAC (Stable Isotope Labeling by Amino Acids in Cell Culture) is a mass spectrometry-based technique used for quantitative proteomics analysis. It enables the relative quantification of proteins in different cell populations by incorporating stable isotope-labeled amino acids into cellular proteins during cell growth and division.

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SILAC medium (9 citations) SILAC DMEM Flex Media (6 citations) RPMI 1640 medium for SILAC (4 citations) SILAC RPMI 1640 medium (2 citations) SILAC FBS (2 citations) SILAC Protein Quantitation kit-DMEM (2 citations) DMEM:F12 for SILAC (2 citations) L-Arginine deficient SILAC Dulbecco's modified Eagle's medium (DMEM) (2 citations) SILAC labeling reagents (2 citations) SILAC Advanced DMEM/F-12 (2 citations)

13 protocols using silac

SILAC Isotopic Labeling for Quantitative Proteomics

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The media utilized for isotopic labeling was Eagle’s minimum essential medium (ATCC) supplemented with 15% dialyzed FBS (Thermo Scientific), 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were gradually adapted to this media by replacing normal FBS with dialyzed FBS within four passages. Cells were then plated at a density of 500,000 cells per 10-cm plate.
One day after plating, the dividing cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with l-arginine:HCl (¹³C6, 99%) and l-lysine:2HCl (¹³C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/L and 0.087 g/L and 15% dialyzed FBS (Thermo Scientific). Cells were collected after 0, 1, 2, and 3 d of labeling and washed with PBS, and cell pellets were frozen before further analysis. Eight days after plating, the confluent quiescent cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with l-arginine:HCl (¹³C6, 99%) and l-lysine:2HCl (¹³C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/L and 0.087 g/L and 15% dialyzed FBS (Thermo Scientific). Cells were collected after 0, 2, 4, and 6 d of labeling and washed with PBS, and cell pellets were frozen before further analysis.

Zhang T., Wolfe C., Pierle A., Welle K.A., Hryhorenko J.R, & Ghaemmaghami S. (2017). Proteome-wide modulation of degradation dynamics in response to growth arrest. Proceedings of the National Academy of Sciences of the United States of America, 114(48), E10329-E10338.

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Cell Culture and Isotopic Labeling for Proteomics

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DU145 and PC3 prostate cancer cell lines were cultured in RPMI 1640 medium (Gibco) supplemented with 10% fetal bovine serum (FBS; Sigma) and 1% penicillin–streptomycin (Corning). HeLa and 293T cell lines were cultured in Dulbecco’s modified Eagle’s medium (Gibco) supplemented with 10% FBS (Sigma) and penicillin–streptomycin (Corning). For SILAC labeling, the DU145 cells were maintained in RPMI 1640 medium for SILAC (Thermo), which was supplemented with 10% dialyzed FBS (Gibco), 1% penicillin–streptomycin, 25 mg/500 ml proline, and 50 mg/500 ml l-arginine and l-lysine (light) or ¹³C₆¹⁵N₄-l-arginine and ¹³C₆¹⁵N₂-l-lysine (heavy). The cells were labeled, respectively, in the light and heavy media for >6 generations before doing a further treatment. For hypoxia treatment, the cells were cultured in an incubator subchamber supplied with 1% O₂/5% CO₂/95% N₂ and monitored by an oxygen sensor (BioSperix). The main chemicals used in this study included puromycin (EMD Millipore), MG132 (APExBIO), dimethyloxalylglycine (DMOG; APExBIO) and deferoxamine (DFO; Cayman).

Luo A., Gong Y., Kim H, & Chen Y. (2020). Proteome dynamics analysis identifies functional roles of SDE2 and hypoxia in DNA damage response in prostate cancer cells. NAR Cancer, 2(2), zcaa010.

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Protein Synthesis Dynamics during Spore Revival

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This approach (Fig. 6D) was used for the experimental analysis of newly synthesized proteins during spore revival. The B. subtilis¹⁴N-labeled spores were germinated in MOPS minimal medium with l-lysine ¹³C₆¹⁵N₂ (Thermo) (210 mg/liter) and l-arginine ¹³C₆¹⁵N₄ hydrochloride (Silantes [here referred as SILAC]) (Thermo) (365 mg/liter) along with a mixture of AGFK and l-alanine as described previously. Samples were taken at t = 0, 15, 30, 60, 90, 150, 210, and 330 min. Here, samples were not taken at the time point before heat activation, i.e., t = −30 min.

Swarge B., Abhyankar W., Jonker M., Hoefsloot H., Kramer G., Setlow P., Brul S, & de Koning L.J. (2020). Integrative Analysis of Proteome and Transcriptome Dynamics during Bacillus subtilis Spore Revival. mSphere, 5(4), e00463-20.

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SIRT6 Turnover Kinetics in HEK293 Cells

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Turnover experiments were performed using HEK293 cell lines stably expressing SIRT6 WT or centSIRT6. Cells were cultured for one week until confluent in MEM for SILAC (Thermo) supplemented with 10% dialyzed FBS (Gibco), L‐glutamine, L‐arginine ¹³C₆¹⁵N₄ (Cambridge Isotopes), and L‐lysine ¹³C₆¹⁵N₂ (Cambridge Isotopes). The media was changed to a normal culture medium, and cell pellets were harvested at 0, 2, 4, 6, 8, 12, and 24 h postmedia change. Cell pellets were lysed in buffer containing 8 M urea, 75 mM NaCl, 50 mM Tris, pH 8.5, with a protease inhibitor cocktail (Roche). Pellets were vortexed for 30s and sonicated 5x 10 s with 1 min rest on ice in between sonication steps. The lysate was centrifuged at 15,000 xg for 10 min, and then, the supernatant was collected.

Simon M., Yang J., Gigas J., Earley E.J., Hillpot E., Zhang L., Zagorulya M., Tombline G., Gilbert M., Yuen S.L., Pope A., Van Meter M., Emmrich S., Firsanov D., Athreya A., Biashad S.A., Han J., Ryu S., Tare A., Zhu Y., Hudgins A., Atzmon G., Barzilai N., Wolfe A., Moody K., Garcia B.A., Thomas D.D., Robbins P.D., Vijg J., Seluanov A., Suh Y, & Gorbunova V. (2022). A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A. The EMBO Journal, 41(21), e110393.

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Stable Isotopic Labeling in Cell Culture

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Before isotopic labeling, cultures were grown to confluency. Once cells ceased cell division and were contact inhibited, they were maintained in a quiescent state for 4 days. Subsequently, cells were acclimated to the labeling media (EMEM) supplemented with 15% dialyzed FBS (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin for 4 days. After 4 additional days in the adaptation media, cultures were introduced to the minimum essential medium for stable isotopic labeling in cell culture (SILAC) (Thermo Scientific) supplemented with L-arginine:HCl (¹³C6, 99%) and L-lysine:2HCl (¹³C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.13 g/l and 0.0904 g/l, respectively, 15% dialyzed FBS (Thermo Scientific), 100 U/ml penicillin, and 100 U/ml streptomycin. After 0, 2, 4, and 6 days of labeling, cells were harvested and washed with PBS, and cell pellets were frozen before further analysis.

Swovick K., Firsanov D., Welle K.A., Hryhorenko J.R., Wise JP S.r., George C., Sformo T.L., Seluanov A., Gorbunova V, & Ghaemmaghami S. (2021). Interspecies Differences in Proteome Turnover Kinetics Are Correlated With Life Spans and Energetic Demands. Molecular & Cellular Proteomics : MCP, 20, 100041.

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SILAC Analysis of Neuronal Globin Interactome

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Stably transfected hNgb-EGFP and EGFP control SH-SY5Y cells were grown in the complete growth medium. For SILAC (stable isotope labeling with amino acid in cell cultures) experiments, the hNgb-EGFP SH-SY5Y cells were maintained in DMEM:F12 (1:1) medium for SILAC (Thermo Fisher Scientific), supplemented with 2 mM L-glutamine (Gibco), 10% dialyzed, heat-inactivated fetal bovine serum (Sigma-Aldrich), and 100 U/ml penicillin-streptomycin (Gibco). L-arginine (Arg0) and L-lysine (Lys0) (‘Light’), ¹³C₆¹⁴N₄-L-arginine (Arg6) and 4,4,5,5-D4-L-lysine (Lys4) (‘Medium’), or ¹³C₆¹⁵N₄-L-arginine (Arg10) and ¹³C₆¹⁵N₂-L-lysine (Lys8) (‘Heavy’) were used for metabolic labeling (0.1 g/L, Thermo Fisher Scientific). In the “reverse” experiment, the labels were moved to the next condition in line as compared to the first run. For the analysis of hNgb-protein interactions under ferroptosis, 50 μM of erastin (Selleckchem) was added to the medium of the labeled cells for the last 24 h of culture.

Van Acker Z.P., Van Raemdonck G.A., Logie E., Van Acker S.I., Baggerman G., Vanden Berghe W., Ponsaerts P, & Dewilde S. (2019). Connecting the Dots in the Neuroglobin-Protein Interaction Network of an Unstressed and Ferroptotic Cell Death Neuroblastoma Model. Cells, 8(8), 873.

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Quantifying UCP1 Dynamics in Adipocytes

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Eight days differentiated adipocyte cells were grown in DMEM medium for SILAC (# 88364, ThermoFisher scientific) supplemented with 10% dialyzed FBS (# 26400044) and amino acid with isotope-labeled ¹³C₆¹⁵N₂-L-Lysine.2HCL (# 88209) / ¹³C₆¹⁵N₄-L-arginine.HCL (# 89990) for 48 h, then washed three times with PBS then cultured with regular DMEM medium supplemented with 10% FBS. The fold change of the log2 ratio between UCP1 protein labeled with heavy-amino acids and UCP1 protein labeled with light-amino acids was determined at day 12 in comparison to day 11.

Yu X., Benitez G., Wei P.T., Krylova S.V., Song Z., Liu L., Zhang M., Xiaoli A.M., Wei H., Chen F., Sidoli S., Yang F., Shinoda K., Pessin J.E, & Feng D. (2024). Involution of brown adipose tissue through a Syntaxin 4 dependent pyroptosis pathway. Nature Communications, 15, 2856.

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Isotopic Labeling Procedure for Proteome Analysis

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The isotopic labeling procedure was similar to that previously described by Zhang et al.^{35 (link)} The media utilized for isotopic labeling was Eagle’s Minimum Essential Medium (ATCC) supplemented with 15% dialyzed FBS (Thermo Scientific), 100 U/mL penicillin, and 100 U/mL streptomycin. Cells were gradually adapted to this media by replacing normal FBS with dialyzed FBS within five passages. Cells were then plated at a density of 1,000,000 cells per 10-cm plate.
One day after plating, the dividing cultures were switched to MEM labeling media for SILAC (Thermo Scientific) supplemented with l-arginine:HCl (¹³C6, 99%) and l-lysine:2HCl (¹³C6, 99%; Cambridge Isotope Laboratories) at concentrations of 0.1264 g/L and 0.087 g/L, 15% dialyzed FBS (Thermo Scientific), 100 U/mL penicillin, and 100 U/mL streptomycin. For whole proteome analysis, cells were collected after 0, 1, 2, 3, and 5 days of labeling and washed with PBS. For analysis of isolated PrP^Sc aggregates, cells were collected after 0, 1, 2, 3, and 4 days of labeling and washed with PBS. All cell pellets were frozen before further analysis.

Hutti C.R., Welle K.A., Hryhorenko J.R, & Ghaemmaghami S. (2020). Global analysis of protein degradation in prion infected cells. Scientific Reports, 10, 10800.

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Metabolite Extraction from Cells

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Cells were re-suspended into RPMI 1640 (Sigma) supplemented with 10% v/v stem cell screened, dialysed fetal bovine serum for SILAC (Thermo Scientific) and 2 mM L-glutamine and seeded into 24-well plates at 0.75x10⁶ cells/ml. Cells were immediately exposed to AZD3965 or DMSO vehicle control. At the appropriate time points the contents of each well were transferred to an eppendorf tube and centrifuged at 13,000 rpm for 2 min. 100 μl media supernatant was aliquoted into an eppendorf tube and proteins were immediately precipitated with 400 μl ice cold 50:50 acetonitrile:methanol for 5 min. Following centrifugation at 13,000 rpm for 5 min the clarified supernatant was then transferred to an eppendorf tube and stored at -20°C until analysis. The remaining media supernatant was discarded and the cell pellet immediately quenched with 400 μl ice cold 40:40:20 acetonitrile:methanol:water for 5 min. The eppendorf tubes were then centrifuged at 13,000 rpm for 5 min to pellet precipitated proteins and cell debris. Clear supernatants were transferred to clear eppendorf tubes and stored at -20°C until analysis.

Curtis N.J., Mooney L., Hopcroft L., Michopoulos F., Whalley N., Zhong H., Murray C., Logie A., Revill M., Byth K.F., Benjamin A.D., Firth M.A., Green S., Smith P.D, & Critchlow S.E. (2017). Pre-clinical pharmacology of AZD3965, a selective inhibitor of MCT1: DLBCL, NHL and Burkitt’s lymphoma anti-tumor activity. Oncotarget, 8(41), 69219-69236.

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Isotope-Labeled Amino Acid Culture Protocol

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Penicillin/streptomycin and Dulbecco’s modified eagle’s medium (DMEM) were obtained from Mediatech (Manassas, VA, USA). SILAC (stable isotope labeling of amino acids in cell culture) media was acquired from Thermo Scientific (Waltham, MA, USA), and fetal bovine serum (FBS), dialyzed FBS for SILAC experiments, and cosmic calf serum (CCS) were purchased from Hyclone (Logan, UT, USA). Unlabeled l-arginine (¹²C₆, ¹⁴N₄) and l-lysine (¹²C₆, ¹⁴N₂) were obtained from MP Biomedicals (Santa Ana, CA, USA), and heavy isotope-labeled l-arginine (¹³C₆, ¹⁵N₄) and l-lysine (¹³C₆, ¹⁵N₂) was purchased from Cambridge Isotope Laboratories, Inc. (Tewksbury, MA, USA). Packing material used for HPLC (high-performance liquid chromatography) was 5 μm C18-coated silica beads, 200 Å pore size, purchased from Michrom Bioresources, Inc. (Auburn, CA, USA). All additional reagents were purchased from Sigma (St. Louis, MO, USA), unless otherwise noted.

Schmoker A.M., Barritt S.A., Weir M.E., Mann J.E., Hogan T.C., Ballif B.A, & Deming P.B. (2018). Fyn Regulates Binding Partners of Cyclic-AMP Dependent Protein Kinase A. Proteomes, 6(4), 37.

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