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.

  • 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 new varieties of heavy amino acids
  • 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

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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.

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.

Comparison of A549 protein levels detected by Western blotting after camptothecin treatment.

Comparison of A549 protein levels detected by Western blotting after camptothecin treatment. Ten micrograms of each light (L) and heavy (H) sample were analyzed by 4-20% SDS-PAGE and Western blotting using specific antibodies.