For SILAC experiments, human endometrial cancer cells Ishikawa were cultured in DMEM/F12 media deficient in L-arginine and L-lysine and supplemented with 10% dialyzed fetal bovine serum (FBS) (Sigma-Aldrich), penicillin, streptomycin, and L-glutamine (Invitrogen) and either L-arginine (Arg-0) and L-lysine (Lys-0), L-arginine [13C6]HCl (Arg-6) and L-lysine-4,4,5,5-d4 (Lys-4), or [13C6, 15N4]HCl (Arg-10) and L-lysine [13C6,15N2]HCl (Lys-8) (Sigma-Aldrich) for 14 days (10 doublings). All media were supplemented with L-proline to prevent the conversion of arginine to proline38 (link). Specifically, isogenic cell lines expressing either vector control (C), wild type SPOP (SPOP-WT) or mutants (MTs) were isotopically labeled with SILAC media and grouped into four experiments (Supplementary Fig. 1d ). Each experiment included a cell line with over-expression of SPOP-WT for cell line comparison within and across experiments. The labeling for this cell line was switched to rule out labeling artifacts in the first three experiments. Approximately 100 million cells per condition were washed twice with PBS, harvested, and snap frozen.
Lys 4
Manufactured by Merck Group
Lys-4 is a laboratory equipment product offered by the Merck Group. It is designed for the purpose of facilitating the analysis and processing of biological samples. The core function of Lys-4 is to assist in the preparation and handling of samples, but a detailed description cannot be provided while maintaining an unbiased and factual approach.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin, by Thermo Fisher Scientific (2 mentions)
Streptomycin, by Thermo Fisher Scientific (2 mentions)
Fetal bovine serum (fbs), by Merck Group (2 mentions)
L glutamine, by Thermo Fisher Scientific (2 mentions)
Gsk591, by Merck Group (1 mentions)
Ms023, by Cayman Chemical (1 mentions)
Silac dmem, by Thermo Fisher Scientific (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
3 protocols using lys 4
1
SILAC-based Proteomics of Endometrial Cancer
2
SILAC-based Proteomics of Endometrial Cancer
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For SILAC experiments, human endometrial cancer cells Ishikawa were cultured in DMEM/F12 media deficient in L-arginine and L-lysine and supplemented with 10% dialyzed fetal bovine serum (FBS) (Sigma-Aldrich), penicillin, streptomycin, and L-glutamine (Invitrogen) and either L-arginine (Arg-0) and L-lysine (Lys-0), L-arginine [13C6]HCl (Arg-6) and L-lysine-4,4,5,5-d4 (Lys-4), or [13C6, 15N4]HCl (Arg-10) and L-lysine [13C6,15N2]HCl (Lys-8) (Sigma-Aldrich) for 14 days (10 doublings). All media were supplemented with L-proline to prevent the conversion of arginine to proline38 (link). Specifically, isogenic cell lines expressing either vector control (C), wild type SPOP (SPOP-WT) or mutants (MTs) were isotopically labeled with SILAC media and grouped into four experiments (Supplementary Fig. 1d ). Each experiment included a cell line with over-expression of SPOP-WT for cell line comparison within and across experiments. The labeling for this cell line was switched to rule out labeling artifacts in the first three experiments. Approximately 100 million cells per condition were washed twice with PBS, harvested, and snap frozen.
3
SILAC Proteomic Analysis of HeLa Cell Lines
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HeLa cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) already including 1% glutamine and supplemented with 10% fetal bovine serum (FBS; Life Technologies), penicillin (100 U/ml), and streptomycin (100 mg/ml). Cells were cultured at 37°C in a 5% CO2 humidified atmosphere. The cells were tested free of mycoplasma contamination. MS023 was purchased from Cayman chemicals; GSK591 was purchased from Sigma Aldrich. Both MS023 (10 µM) and GSK591 (5 µM) were used for 48 h treatment, together with DMSO as control. For triple SILAC, HeLa were grown in ‘‘Light’’, ‘‘Medium’’ and ‘‘Heavy’’ SILAC DMEM (Thermo Fisher Scientific), supplemented with either L-Arginine, L-Lysine or their medium (Arg6: Sigma-Aldrich; Lys4: Sigma-Aldrich) or heavy (Arg10: Sigma-Aldrich; Lys8: Sigma-Aldrich) isotope-counterparts. Arginine and Lysine were added at a concentration of 84 mg/L and 146 mg/L, respectively. SILAC media were supplemented with 10% dialyzed FBS (GIBCO, Life Technologies), 100 U/ml Penicillin and 100 mg/ml Streptomycin. HeLa cells were grown in the respective heavy-isotopes containing media for at least 9 replication cycles, to ensure full incorporation of isotope-encoded amino acids, with a careful monitoring of growth rate, viability and overall morphology, to guarantee that normal cell physiology was preserved.
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