SILAC reagents were purchased from Thermo Fisher with the exception of FCS and SILAC DMEM from Sigma. RPE1 cells virally expressing selected plasmids were seeded in six-well plates in SILAC labelling medium supplemented with dialysed FCS and cultured over a minimum of six passages to achieve full labelling with respective isotopes and a minimum of two confluent 20 cm dishes for generation of lysates. GFP-expressing control cells were grown in unlabelled medium with standard arginine and lysine (R0K0) and GFP-SNX21-expressing cells were grown in medium supplemented with ‘medium’-mass isotopes [13C6]-arginine and 4,4,5,5-D4-Lysine (R6K4). Cells were lysed in immunoprecipitation buffer [50 mM Tris-HCl (pH 7.4), 0.5% NP40, 1 mM PMSF, 200 µM Na3VO4 and a Roche mini complete protease inhibitor tablet] and the GFP tags were precipitated with GFP-nanotrap beads (Chromotek) for 1 h at 4°C then combined prior to three washes in wash buffer (50 mM Tris-HCl, pH7.4, 0.2% NP40). Proteins were isolated in sample buffer, separated using NuPAGE (4-12%) pre cast gels (Invitrogen), visualised using All Blue protein stain (Invitrogen) and analysed by LC-MS-MS on an Orbitrap Velos (Thermo) spectrophotometer (Steinberg et al., 2012 (link); Steinberg et al., 2013 (link); McGough et al., 2014a (link),b (link); McMillan et al., 2016 (link)).
Silac dmem
Manufactured by Merck Group
SILAC DMEM is a powdered cell culture media designed for Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) experiments. It is available in both unlabeled and labeled forms to facilitate comparative proteomic studies.
Automatically generated - may contain errors
Lab products found in correlation
L glutamine, by Merck Group (2 mentions)
Silac dmem, by Thermo Fisher Scientific (2 mentions)
Orbitrap velos, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Merck Group (1 mentions)
Nupage 4 12 precast gels, by Thermo Fisher Scientific (1 mentions)
Mini complete protease inhibitor tablet, by Proteintech (1 mentions)
Gfp nanotrap beads, by Proteintech (1 mentions)
Silac reagents, by Thermo Fisher Scientific (1 mentions)
L arginine hcl, by Thermo Fisher Scientific (1 mentions)
T175 culture flask, by Sarstedt (1 mentions)
L glutamine, by Cambridge Isotopes (1 mentions)
G8796, by Merck Group (1 mentions)
C7629, by Merck Group (1 mentions)
L8912, by Merck Group (1 mentions)
L8662, by Merck Group (1 mentions)
A8094, by Merck Group (1 mentions)
A5131, by Merck Group (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Non essential amino acids (neaa), by Thermo Fisher Scientific (1 mentions)
Pyruvate, by Thermo Fisher Scientific (1 mentions)
6 protocols using silac dmem
1
Quantitative Proteomic Analysis of SNX21
2
SILAC-based Proteomic Analysis of C. jejuni
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C. jejuni strain M1 and its isogenic flgG mutant were grown on 1% SILAC DMEM plates supplemented with 10 mM l -Glutamine (Sigma) and either l -Arginine-HCL (Thermo Fisher Scientific) or l -Arginine 13C6, 15N4 (Thermo Fisher Scientific). Powdered SILAC DMEM (Thermo Fisher Scientific) was dissolved in water to create a 2 × SILAC DMEM solution. Amounts of either l -Arginine-HCL or l -Arginine 13C6, 15N4 were added to the solution, dissolved, and passed through a 0.22 μm filter. To this, an equal volume of sterile 2% select agar (Sigma) was added and supplemented with 10 mM l -Glutamine. For validation of the SILAC data by Western immunoblotting, 1% DMEM plates were composed of 2 × standard DMEM (Millipore) mixed with 2% sterile agar as above, and supplemented with 10 mM l -Glutamine. Bacterial strains were streaked on relevant media from frozen stocks and incubated at 42 °C under microaerophilic conditions for 48 h.
3
Cultivation and SILAC Labeling of HeLa Cells
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Human
epithelioid cervix carcinoma HeLa
cells were purchased from Sigma-Aldrich (93021013_1VL) and cultivated
in a T175 culture flask (Sarstedt) containing high glucose Dulbecco’s
Modified Eagle’s Medium (DMEM) (Sigma-Aldrich) supplemented
with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich)
and 2 mMl -glutamine (Sigma-Aldrich). Cells were maintained
at 37 °C in a humidified 5% CO2 atmosphere. The cells
were routinely tested for mycoplasma contamination.
For SILAC
experiments, HeLa cells were passaged at least six times in SILAC-DMEM
(Sigma-Aldrich) supplemented with 10% dialyzed FBS and 2 mMl -glutamine as well as 214 μM [13C6, 15N4] l -arginine HCl (Arg10) and 419 μM
[13C6, 15N2]l -lysine 2 HCl (Lys8) (Cambridge Isotope Laboratories) resulting in
“heavy” cells or with 214 μM [13C6]l -arginine HCl (Arg6) and 419 μM [4,4,5,5-D4] l -lysine 2 HCl (Lys4) (Cambridge Isotope Laboratories) resulting
in “light” cells instead.
epithelioid cervix carcinoma HeLa
cells were purchased from Sigma-Aldrich (93021013_1VL) and cultivated
in a T175 culture flask (Sarstedt) containing high glucose Dulbecco’s
Modified Eagle’s Medium (DMEM) (Sigma-Aldrich) supplemented
with 10% heat-inactivated fetal bovine serum (FBS) (Sigma-Aldrich)
and 2 mM
at 37 °C in a humidified 5% CO2 atmosphere. The cells
were routinely tested for mycoplasma contamination.
For SILAC
experiments, HeLa cells were passaged at least six times in SILAC-DMEM
(Sigma-Aldrich) supplemented with 10% dialyzed FBS and 2 mM
[13C6, 15N2]
“heavy” cells or with 214 μM [13C6]
in “light” cells instead.
4
Arginine-free Complete DMEM and RPMI Preparation
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Complete Dulbecco’s Modified Eagles Media (C-DMEM) was prepared by adding bovine calf serum (SH30073.03, Thermo Fisher Scientific) to 10%, and penicillin/streptomycin (15140-122, Gibco, Life Technologies) to 1% in standard DMEM (10-013-CV, Cellgro, Corning Life Sciences). L-arginine-free (R-free) C-DMEM lacking phenol red was prepared by adding dialyzed fetal bovine serum (35-071-CV, Cellgro, Corning Life Sciences) to 10% in SILAC DMEM (D9443, Sigma-Aldrich) plus L-glutamine (584 mg/L, 25030-081, Life Technologies), L-lysine-HCl (146.2 mg/L, L8662, Sigma-Aldrich), L-leucine (52.5 mg/L, L8912, Sigma-Aldrich), sodium pyruvate (110 mg/L, 11360-070, Life Technologies), D-glucose (4500 mg/L, G8796, Sigma-Aldrich) and phenol red (15 mg/L p3532, Sigma-Aldrich). For luminescence experiments, phenol red was excluded from cell culture media. L-arginine (A8094, Sigma-Aldrich), L-citrulline (C7629, Sigma-Aldrich), and L-arginine · HCl (A5131, Sigma-Aldrich) were prepared at a stock concentration of 100 mM in sterile water. L-arginine and L-citrulline stocks were added to R-free C-DMEM to concentrations noted in the text. Complete Roswell Park Memorial Institute 1640, (C- RPMI) was prepared by adding dialyzed fetal bovine serum to 10% and penicillin/streptomycin to 1% in standard RPMI 1640 (10-040-CV, Corning Life Sciences).
5
Cell Culture and SILAC Labeling
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HeLa, HEK293-T and RPE1 cells were cultured at 37°C and 5% CO2 in DMEM supplemented with 10% FCS and 1% penicillin-streptomycin. For SILAC experiments, RPE1 cells were cultured with labelled amino acids (R6K4 or R0K0) contained in SILAC-DMEM (Sigma) supplemented with 10% dialysed FCS (Sigma).
6
SILAC-Based Proteomic Analysis of Cell Lines
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Cell lines SH-SY5Y and T-REx-293 cells were cultured under standard cell culture conditions. In brief, cells were cultured in DMEM (Life Technologies) complemented with 10% fetal calf serum (Pan-Biotech). Cells used for SILAC based experiments were cultured in SILAC DMEM (Life Technologies) complemented with glutamine (Glutamax, Life Technologies), Pyruvate (Life Technologies), non-essential amino acids (Life Technologies) and 10% dialyzed fetal calf serum (Pan-Biotech). The SILAC DMEM was supplemented with standard L-arginine (Arg0, Sigma-Aldrich) and L-lysine (Lys0, Sigma-Aldrich) (''light'') as in Schwanha ¨usser et al. (2011) . Alternatively, Arg6 and Lys4 (''medium-heavy'') or Arg10 and Lys8 (''heavy'') were added in place of their light counterparts. Cells were cultured at 37 C and 5% CO 2 .
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