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