產品名稱：SILAC Protein Quantitation  Kit-RPMI 1640 Kit
貨號：Pierce 89982
規格：EACH
Product Details:
    Example SILAC workflow. Stable isotope labeling with amino acids in cell culture （SILAC） requires growing mammalian cells in specialized media deficient in lysine and arginine. This deficiency is compensated by adding light or heavy forms of the missing amino acids; i.e., 12C6 and 13C6 L-lysine, respectively. A typical experiment involves growing one cell population in medium containing light amino acids （control）, while the other population is grown in the presence of heavy amino acids （experimental）. The heavy and light amino acids are incorporated into proteins through natural cellular protein synthesis. After alteration of the proteome in one sample through chemical treatment or genetic manipulation, equal amounts of protein from both cell populations are then combined, separated by SDS polyacrylamide gel electrophoresis （SDS-PAGE） and digested with trypsin before MS analysis. 
Because peptides labeled with heavy and light amino acids are chemically identical, they co-elute during reverse-phase column pre-fractionation and therefore are detected simultaneously during MS analysis. The relative peak intensities of multiple isotopically distinct peptides from each protein are then used to determine the average change in protein abundance in the treated sample.
Each kit includes all necessary reagents to isotopically label cells with 13C6 L-lysine-2HCl, including media, heavy and light amino acids and dialyzed serum. Heavy L-arginine-HCl derivatives （Part No. 88210, 89990） are available separay and can be combined with Pierce SILAC Protein Quantitation Kits to enhance peptide isotope label coverage. Kits are compatible with mammalian cell lines adapted to grow in either DMEM or RPMI 1640 media. Kits with specialized media formulations are also available for human and murine stem cell lines. When combined with Thermo Scientific Protein/Peptide Sample Enrichment Products, Pierce SILAC Protein Quantitation Kits also enable MS analysis of low-abundance proteins such as cell surface proteins, organelle-specific proteins and post-translational protein modifications such as phosphorylation or glycosylation.
Example Data:
Using a Pierce SILAC Quantitation Kit, A549 cells adapted to DMEM were grown for six passages （10 days） using SILAC DMEM containing 0.1 mg/ml heavy 13C6 L-lysine-2HCl or light L-lysine-2HCl supplemented with 10% dialyzed FBS. After * label incorporation, heavy-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 Reagent （Part No. 78501）. Samples were normalized for protein concentration using the Thermo Scientific Pierce BCA Protein Assay （Part No. 23225）, and 50 μg of each sample was equally mixed and analyzed by 4-20% SDS-PAGE. Gels were stained with Thermo Scientific GelCode Blue Stain Reagent （Part No. 24592）, and proteins were digested and alkylated using the Thermo Scientific Pierce In-Gel Tryptic Digestion Kit （Part No. 89871） before analysis using an LTQ Orbitrap Hybrid Mass Spectrometer.
Overall, more than 350 proteins were successfully identified by MS/MS sequencing using a Thermo Scientific LTQ Orbitrap Mass Spectrometer. Identified peptides were then quantitated using the Thermo Scientific Proteome Discoverer Software Suite to generate SILAC ratios corresponding to relative changes in protein abundance. Most of the proteins identified showed no change in abundance after camptothecin treatment （Figure）; however, 13% of proteins quantified in heavy-labeled cells had protein levels （SILAC ratios） 1.5-fold higher than in control cells. More than 6% of proteins showed a reduced relative abundance by the same amount after drug treatment, while 15 proteins were identified by MS as those most significantly up-regulated after drug treatment （Table）. One protein that was identified as being up-regulated in response to camptothecin treatment was proliferating cell nuclear antigen （PCNA）, a protein known to be involved in DNA repair （Figure）. To validate the SILAC data, protein levels were separay quantitated by Western blot （Figure）. PCNA protein levels increased 1.9-fold compared to those of the housekeeping protein glyceraldehyde-3-phosphate dehydrogenase （GAPDH）. The abundance ratios determined by Western blot were comparable to those determined by SILAC （Table）.