A Practical Formulation Guide for SILAC Technology

What Is SILAC Technology?

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC) technology involves the metabolic incorporation of stable isotope-labeled amino acids into cellular proteins during cell culture. SILAC is a simple, stable and powerful method in quantitative proteomics based on mass spectrometry (MS). In quantitative proteomics, SILAC can provide accurate relative quantification without any chemical derivatization or other operations, and is widely used in research fields such as cell biology, biochemistry, and pharmacology.

SILAC Medium Formulation

The formulation of SILAC media is a critical step in implementing this technology effectively. The SILAC medium should contain all essential amino acids, with one or more of them replaced with the corresponding heavy isotope-labeled amino acid.

Light medium
Normal culture medium such as DMEM (Dulbecco's Modified Eagle Medium), RPMI 1640 (Roswell Park Memorial Institute), and MEM (Minimum Essential Medium).
Heavy Medium
Use ¹³C or ¹⁵N-labeled amino acids, such as leucine (¹³C-Leu), lysine (¹³C-Lys) and methionine (¹³C-Met).
Other ingredients
Include other nutrients required for cell growth, such as dialyzed fetal bovine serum and specific supplements.

Fig 1. Overview of SILAC experiment. Fig 1. SILAC protocol. [1]

Cell culture
The cells are divided into two parts, one part is cultured in light-labeled medium and the other part is cultured in heavy-labeled medium. After 6-7 doubling cycles, the amino acids labeled with stable isotopes in cells cultured in heavy-labeled medium can be completely incorporated into newly synthesized proteins.

Notes
Labeling efficiency detection: It is recommended to perform labeling efficiency detection after the cells have undergone multiple doubling cycles to ensure that the labeling efficiency meets the SILAC technical requirements.
Experimental operation: In order to more conveniently perform drug treatment or conditional stimulation, it is recommended to perform experiments in light-labeled cultured cells.

For more details, please see our SILAC service.

Choice of Isotope-Labeled Amino Acids in SILAC

The choice of isotope-labeled amino acids in SILAC technology is crucial for accurate and specific protein labeling. Popular choices include heavy isotope-labeled forms of lysine (¹³C₆, ¹⁵N₂-lysine) and arginine (¹³C₆, ¹⁵N₄-arginine), which can be incorporated into newly synthesized proteins during cell culture. The following table lists the amino acid composition and quality differences used in SILAC culture media.

Light Amino Acids	Heavy Amino Acids	Heavy vs. Light
Name	Abbreviation	Name	Abbreviation	Dalton
Lysine	K0	L-Lysine-13C6 dihydrochloride	K6	6 Da
L-Lysine-d4 dihydrochloride	K4	4 Da
L-Lysine-d4 hydrochloride	K4	4 Da
L-Lysine-13C6, 15N2 dihydrochloride	K8	8 Da
L-Lysine-15N2 hydrochloride	K2	2 Da
Arginine	R0	L-Arginine-13C6 hydrochloride	R6	6 Da
L-Arginine-13C6, 15N4 hydrochloride	R10	10 Da
L-Arginine-15N4 hydrochloride	R4	4 Da
L-Arginine-15N2 hydrochloride	R2	2 Da
Leucine	L0	L-Leucine-d10	L10	10 Da
L-Leucine-1-13C, 15N	L2	2 Da
L-Leucine-d3	L3	3 Da
L-Proline	P0	L-Proline-13C5	P5	5 Da
L-Proline-15N	P1	1 Da
L-Methionine	M0	L-Methionine-13C5	M5	5 Da
L-Methionine-methyl-13C	M1	1 Da
L-Methionine-d3	M3	3 Da

Light Amino Acids

Heavy Amino Acids

Heavy vs. Light

Name

Abbreviation

Name

Abbreviation

Dalton

Lysine

L-Lysine-13C6 dihydrochloride

6 Da

L-Lysine-d4 dihydrochloride