SILAC Metabolic Labeling Systems

Stable isotope labeling using amino acids in cell culture (SILAC) is a powerful method to identify and quantify relative differential changes in complex protein samples. The SILAC method uses in vivo metabolic incorporation of “heavy” 13C- or 15N-labeled amino acids into proteins followed by mass spectrometry (MS) analysis for accelerated comprehensive identification, characterization and quantitation of proteins. NeuCode amino acids enable up to four samples to be multiplexed simultaneously.

  • Reproducible—eliminates intra-experimental variability caused by differential sample preparation
  • Flexible—media deficient in both L-lysine and L-arginine, as well as Flex media formulations that allow the addition of glucose, glutamine and phenol red separately
  • Versatile—broadest portfolio of liquid and powdered SILAC media, as well as unique NeuCode amino acids, enabling higher multiplexing
  • Convenient—media and amino acids are available in kits or as stand-alone reagents
  • Compatible—label proteins expressed in a wide variety of mammalian cell lines, including HeLa, 293T, COS7, U2OS, A549, NIH 3T3, Jurkat and others

How-to video

SILAC metabolic labeling using mass spectrometry

Learn how to prepare and metabolically label cultured cells using standard heavy or NeuCode amino acids for SILAC applications using mass spectrometry.

Ordering information

SILAC kits
NeuCode amino acids
SILAC amino acids
SILAC media and dialyzed FBS

Featured product data

Procedure summary for MS experiments using SILAC reagents. Normalized protein extracts isolated from cells are combined, reduced, alkylated, and digested overnight. For the in-gel workflow, samples are run on an SDS-PAGE gel, excised, digested, and cleaned up; for the in-solution workflow, samples are digested, fractionated, and cleaned up. Samples are then analyzed by high-resolution Orbitrap LC-MS/MS.

Representative MS spectra generated using SILAC.
Representative MS spectra generated using SILAC. Light and heavy (13C6) L-lysine-containing peptides (AEDNADTLALVFEAPNQEK) from PCNA were analyzed by MS. Mass spectra of heavy peptides containing 13C6 L-lysine have an increased mass of 6Da and are shifted to the right of light peptide spectra by a mass to charge ratio (m/z) of 3 caused by a +2 ionization of peptides

Table 1. SILAC isotopes of amino acids available to enable multiplexed experiments and analysis.

Amino acid Light D4 13C6 D8 13C15N2 13C615N4
Mass shift 0 Da +4 Da +6 Da
 +8 Da +8 Da +10 Da
L-Arginine-HCl Cat.No.
89989 (50 mg)
88427 (500 mg)		 N/A Cat.No.
88210 (50 mg)
88433 (500 mg)		 N/A N/A Cat.No.
89990 (50 mg)
88434 (500 mg)
L-Leucine 88428 (500 mg) N/A 88435 (50 mg)
88436 (500 mg)		 N/A N/A N/A
L-Lysine-2HCl 89987 (50 mg)
88429 (500 mg)		 88437 (50 mg)
88438 (500 mg)		 89988 (50 mg)
88431 (500 mg)		 A33613 (50 mg)
A33614 (500 mg)		 88209 (50 mg)
88432 (500 mg)		 N/A
L-Proline 88211 (115 mg)
88430 (500 mg)		 N/A N/A N/A N/A N/A

Table 2. Thermo Scientific NeuCode isotopes of L-lysine-2HCl available to enable multiplex experiments and analysis.

+4 Da +8 Da
Amino acid K202 K040 K080 K521 K341 K440 K602
Mass shift 4.00078 4.02511 8.01420 8.02637 8.03221 8.03853 8.05021
Cat. No. A36754 (25 mg) 88437 (50 mg)
88438 (500 mg)		 A36750 (25 mg)
A33613 (50 mg)
A33614 (500 mg)		 A36753 (25 mg) A36851 (25 mg) A36752 (25 mg) A36751 (25 mg)
88209 (50 mg)
88432 (500 mg)
SILAC workflow

Procedure summary for MS experiments using SILAC reagents. Normalized protein extracts isolated from cells are combined, reduced, alkylated, and digested overnight. For the in-gel workflow, samples are run on an SDS-PAGE gel, excised, digested, and cleaned up; for the in-solution workflow, samples are digested, fractionated, and cleaned up. Samples are then analyzed by high-resolution Orbitrap LC-MS/MS.

Representative MS spectra
Representative MS spectra generated using SILAC.
Representative MS spectra generated using SILAC. Light and heavy (13C6) L-lysine-containing peptides (AEDNADTLALVFEAPNQEK) from PCNA were analyzed by MS. Mass spectra of heavy peptides containing 13C6 L-lysine have an increased mass of 6Da and are shifted to the right of light peptide spectra by a mass to charge ratio (m/z) of 3 caused by a +2 ionization of peptides
SILAC amino acid isotopes

Table 1. SILAC isotopes of amino acids available to enable multiplexed experiments and analysis.

Amino acid Light D4 13C6 D8 13C15N2 13C615N4
Mass shift 0 Da +4 Da +6 Da
 +8 Da +8 Da +10 Da
L-Arginine-HCl Cat.No.
89989 (50 mg)
88427 (500 mg)		 N/A Cat.No.
88210 (50 mg)
88433 (500 mg)		 N/A N/A Cat.No.
89990 (50 mg)
88434 (500 mg)
L-Leucine 88428 (500 mg) N/A 88435 (50 mg)
88436 (500 mg)		 N/A N/A N/A
L-Lysine-2HCl 89987 (50 mg)
88429 (500 mg)		 88437 (50 mg)
88438 (500 mg)		 89988 (50 mg)
88431 (500 mg)		 A33613 (50 mg)
A33614 (500 mg)		 88209 (50 mg)
88432 (500 mg)		 N/A
L-Proline 88211 (115 mg)
88430 (500 mg)		 N/A N/A N/A N/A N/A

Table 2. Thermo Scientific NeuCode isotopes of L-lysine-2HCl available to enable multiplex experiments and analysis.

+4 Da +8 Da
Amino acid K202 K040 K080 K521 K341 K440 K602
Mass shift 4.00078 4.02511 8.01420 8.02637 8.03221 8.03853 8.05021
Cat. No. A36754 (25 mg) 88437 (50 mg)
88438 (500 mg)		 A36750 (25 mg)
A33613 (50 mg)
A33614 (500 mg)		 A36753 (25 mg) A36851 (25 mg) A36752 (25 mg) A36751 (25 mg)
88209 (50 mg)
88432 (500 mg)
NeuCode isotopologes

Different combination of lysine isotopologs can be used to increase multiplexing from 2-plex to 4-plex at high resolution.

Resources

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For Research Use Only. Not for use in diagnostic procedures.