Apr 30, 2026
SILAC DMEM 2 recipe
This protocol is a draft, published without a DOI.
- 1Discovery Research Platform for Hidden Cell Biology
Protocol Citation: Georg Kustatscher 2026. SILAC DMEM 2 recipe. protocols.io https://dx.doi.org/
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: June 21, 2024
Last Modified: April 30, 2026
Protocol Integer ID: 102220
Keywords: dmem for enrichable methionine analogue, methionine labelling media, silac dmem, lysine depletion media, enrichable methionine analogue, precipitates amino acid, abundant peptide, thaw 500ml bottle, recipe media recipe, ml in ultrapure water, using light arginine, dmem, methionine to media aliquot, amino acid, protein gradient, methionine, light arginine, silac, doing silac, lysine, cell culture media, heavy arginine
Abstract
Media recipe for DMEM for enrichable methionine analogue labelling with or without SILAC.
Complete media: make up with all components as specified using light arginine and lysine
Depletion media: do not add arginine, lysine or methionine
Labelling media: add heavy arginine and lysine if doing SILAC, otherwise add light. Do not add methionine. Add AHA or methionine to media aliquot before experiment.
Notes
- Van originally used 50mg/500ml for all RKP but there was a very small amount of R10->P6 conversion visible in MS1 for abundant peptides
- Dialysed FBS: Label with dialysed FBS batch so that L and H media with the same dFBS can be used in experiment. Freeze thaw cycles should be limited, so thaw 500ml bottle and make 50ml aliquots for adding to cell culture media and store at -20°C. Thaw at 4°C or at 37°C, ensuring to mix throughout thawing to prevent salt/protein gradients which increase precipitates
- Amino acids: Make up arginine, lysine and proline to 0.1g/ml in UltraPure water (Invitrogen; 10977035), filter-sterilise using 0.2um filters and aliquot
c
- Deficient in arginine, lysine, methionine, leucine, sodium pyruvate and phenol red
50ml dialysed FBS (Gibco, 26400044)
- Spin 3202g 3 min before adding to remove precipitate
5ml 100X Penicillin-Streptomycin (Gibco 11548876)
5ml 100X Glutamax (Thermo 35050061)
5ml Sodium pyruvate 100mM (Gibco 11360070)
Arginine (Heavy Arginine.HCl: Sigma, 608033 or Light Arginine: Sigma, A8094-100G)
- 250ul heavy from 0.1g/ml stock (25mg/500ml; 50mg/L; 227uM)
- 197.4ul light from 0.1g/ml stock (19.74mg/500ml; 39.485mg/L; 227uM)
Lysine (Heavy Lysine.HCl; Sigma 608041 or Light Lysine.HCl Sigma, L8662-100G)
- 500ul heavy from 0.1g/ml stock (50mg/500ml; 100mg/L; 525uM)
- 479.2ul light from 0.1g/ml stock (47.94mg/500ml; 95.834mg/L; 525uM)
Proline (Sigma, P5607-25G)
- 2ml from 0.1g/ml stock (200mg/500ml; 400mg/L; 3.47mM)
2.5ml leucine (supplied with media)
500ul methionine (supplied with media)