Sample SILAC Recipe

 DMEM with 13C6 Arginine and 13C6 Lysine

Dulbecco's Eagle's minimum essential medium deficient in amino acids arginine and lysine was custom synthesized by AthenaES (Baltimore, MD). Add amino acids L-lysine (0.798 mM) and 13C6 Arginine (0.398 mM) to 20 ml of the custom synthesized medium. The amounts of amino acids to be added to the media are shown in table . After ensuring that all the amino acids have dissolved, filter the medium through a 0.22 m filter to obtain sterile, complete medium before making it up to 1 L media. Before using, supplement the media with antibiotics and 10% fetal bovine serum (FBS). The above recipe can be used in a similar way when RPMI is the propagating medium for the cells where the molar concentrations of the amino acids in the formulation are also listed in table.

Stable isotopic amino acids used in SILAC media
Stable isotopic
amino acids (MW)
Concentration
in DMEM
Concentration
in RPMI
Mass difference Cambridge
Isotope Catalog
Sigma-Aldich
Catalog
L-Lysine 2HCl (223.13)
(4,4,5,5-D4)
178.0 mg/l
(0.798 mM)
61.14 mg/l
(0.274 mM)
4 Da DLM-2640 -
L-Lysine HCl (186.67)
(4,4,5,5-D4)
149.0 mg/l
(0.798 mM)
51.15 mg/l
(0.274 mM)
4 Da - 616192
L-Lysine 2HCl (225.1)
(13C6)
179.6 mg/l
(0.798 mM)
61.68 mg/l
(0.274 mM)
6 Da CLM-2247 -
L-Lysine HCl (188.6)
(13C6)
150.5 mg/l
(0.798 mM)
51.68 mg/l
(0.274 mM)
6 Da - 643459
L-Lysine 2HCl (227.1)
(13C6, 15N2)
181.2 mg/l
(0.798 mM)
62.23 mg/l
(0.274 mM)
8 Da CNLM-291 -
L-Lysine HCl (190.59)
(13C6, 15N2)
152.1 mg/l
(0.798 mM)
52.22 mg/l
(0.274 mM)
8 Da - 608041
L-Arginine HCl (214.64)
(15N4)
85.4 mg/l
(0.398 mM)
246.8 mg/l
(1.15 mM)
4 Da NLM-396 600113
L-Arginine HCl (216.62)
(13C6)
86.2 mg/l
(0.398 mM)
249.1 mg/l
(1.15 mM)
6 Da CLM-2265 643440
L-Arginine HCl (220.59)
(13C6, 15N4)
87.8 mg/l
(0.398 mM)
253.7 mg/l
(1.15 mM)
10 Da CNLM-539 608033
L-Arginine HCl (221.6)
(15N4, D7)
88.2 mg/l
(0.398 mM)
254.8 mg/l
(1.15 mM)
11 Da DNLM-7543 -
L-Arginine HCl (227.6)
(13C6, 15N4, D7)
90.6 mg/l
(0.398 mM)
261.7 mg/l
(1.15 mM)
17 Da CDNLM-6801 -

Notes:

  1. All the above steps should be carried out under a laminar flow hood. Store the media at 4C until use.
  2. The cells are always adapted to the custom SILAC medium for 5 passages to achieve complete incorporation of the isotope labeled amino acid before they are propagated to a scale needed for the experiment.
  3. We have tested many different attached cell lines (NIH3T3, 293T, HeLa, HepG2, 3T3L1) and suspension cell lines (HeLa S3, BaF3, PC-12). We find that SILAC labeling has no deleterious effect on cells in terms of growth and division, morphology, or biological responses.
  4. One can use lower amounts of heavy isotope labeled amino acids in the preparation of custom SILAC media, than are present in the original formulation if the cost of heavy amino acids is a consideration as long as the cells are monitored. However in such cases it is advisable to present the cells with similar amounts of light amino acid.


Lysis Buffer:

  • 150 mM NaCl
  • 50 mM Tris, pH 7.4
  • 1 % NP-40


Modified RIPA Buffer:

  • 150 mM NaCl
  • 50 mM Tris, pH 7.4
  • 1 % NP-40
  • 0.25 % Sodium deoxycholate
  • EDTA 1 mM


Fixation Solution for Silver Staining:

  • Methanol: Acetic acid: Water 50:5:45 (v/v/v)


Sensitizing Solution for Silver Staining:

  • Sodium thiosulfate 0.02%


Staining Solution:

  • Silver nitrate 0.1%


Developing Solution:

  • Formaldehyde 0.04% in 2% sodium carbonate
  • Methanol 50%
  • Formic acid 5%