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.
- 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 specialty kits optimized for phosphoprotein and membrane protein extraction and enrichment
- 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
Choose the right SILAC kit for your application
|Order now||Order now||Order now||Order now|
|SILAC™ Protein Quantitation Kits||SILAC™ Protein ID & Quantitation Media Kit||SILAC™ Phosphoprotein ID & Quantitation Kit||SILAC™ Membrane Protein ID & Quantitation Kit|
|Application||Total protein expression analysis||Total protein expression analysis||Phosphoprotein expression analysis||Membrane protein expression analysis|
|Media configuration||2 x 500 mL||2 x 1 L||2 x 1 L||2 x 1 L|
|Heavy lysine, light L-lysine and L-arginine included?||Yes||Yes||Yes||Yes|
|L-glutamine, glucose and phenol red||Complete formulations||Without glucose and phenol red||Without L-glutamine, glucose, and phenol red||Without L-glutamine, glucose, and phenol red|
|Membrane lysis buffer||No||
|Phosphoprotein lysis buffer and PiMAC™ Resin||No||No||Yes||No|
Need to order amino acids products separately?
Heavy and light amino acids are used to specifically analyze protein expression by mass spectrometry using stable isotope labeling with amino acids in cell culture (SILAC) quantification kits.
Need to order SILAC media or dialyzed FBS separately?
SILAC cell culture media and dialyzed fetal bovine serum (FBS) are optimized for use with stable isotope labeling with amino acids in cell culture (SILAC) to analyze protein expression by mass spectrometry (MS).
Schematic of SILAC workflow. A549 cells adapted to DMEM were grown for six passages (10 days) using SILAC DMEM (Product # 89983) containing 0.1 mg/mL heavy 13C6 L-lysine-2HCl or light L-lysine-HCl supplemented with 10% dialyzed FBS. After 100% label incorporation, 13C6 L-lysine-labeled cells were treated with 5 μM camptothecin for 24 hours. Cells from each sample (light and heavy) were lysed using Thermo Scientific™ M-PER™ Mammalian Protein Extraction Reagent (Product # 78501). Samples were normalized for protein concentration using the Thermo Scientific™ Pierce™ BCA Protein Assay (Product # 23225), and 50 mg of each sample were equally mixed before 4-20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. Gels were stained with Thermo Scientific™ GelCode™ Blue Stain Reagent (Product # 24592) and proteins were digested and alkylated using the Thermo Scientific™ Pierce™ In-Gel Tryptic Digestion Kit (Product # 89871) before analysis using a Thermo Scientific™ LTQ Orbitrap™ Mass Spectrometer.
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. Ten micrograms of each light (L) and heavy (H) sample were analyzed by 4-20% SDS-PAGE and Western blotting using specific antibodies.
For Research Use Only. Not for use in diagnostic procedures.