Metabolic Labeling - SILAC™ Introduction

SILAC™ Protein ID and Quantitation Kits: Your Complete Solution for Differential Protein Expression Analysis

SILAC™ ID and Quantitation labeling kits provide protocols that are compatible with cell culture workflows. By adding stable, non-radioactive isotopic forms of amino acids to media when you grow your cells, you can achieve 100% amino acid incorporation. Once the amino acids are incorporated, you can quantify relative protein abundance as low as 30% with coefficient of variation as low as 8%. Because samples are mixed early on before processing and are subjected to the identical experimental protocol, experimental results achieve high fidelity with minimal bias, allowing relative quantitation of even small changes in protein abundance.
 
Product Attributes:
  • Metabolic labeling method ensuring the highest fidelity
  • 100% label incorporation with labeling complete in six passages
  • Fractionate using standard protein chromatography or gel separation techniques prior to MS analysis
  • Compatible with common cell culture workflows
  • Includes all components for complete experiment including: light and heavy amino acids (Lysine), Gibco brand media, FBS, Glutamine, lysis buffers, etc.

Figure 1. Typical SILAC™ Technology Workflow
Silac Bottles
Step 1
Duplicate Silac
Grow duplicate cultures in SILAC™ media with isotopically distinct amino acids (AA)
Step 2
Silac Apply StimuliIf needed apply stimulus (e.g. RNAi, etc.) to culture grown in SILAC™ Media. Harvest and mix cells.
Step 3
Silac GelLyse cells using SILAC™ lysis buffers "run lysate on NuPAGE™ ID gel", excise gel band and trypsinize.
Step 4
Silac Data AnalysisSample submission to MS operator for data analysis.
Step 5
Silac List ProteinsObtain list of proteins identified with relative abundances.

Figure 2. A single protocol identifies > 1600 proteins without prefractionation

Simple Cell Culture Workflow
Using the SILAC™ Membrane Protein ID and Quantitation Kit without pre-fractionation, more than 1,600 proteins were identified from breast cancer cells, of which 1,000 were membrane proteins and 250 proteins have unknown function. Several classes of proteins were identified, including 350 histones, heat shock proteins, actins, among others. Quantitative analysis identified 150 proteins with at least 2 fold change in expression; 77 proteins with at least 3 fold change in expression; 40 proteins with at least 5 fold change in expression.
SILAC™ Quantitation and Labeling Kits ordering information
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