2D Gel Electrophoresis

2D PAGE and Mass Spectrometric Protein Identification

2D electrophoresis is the classical proteomics method for separating and visualizing complex protein mixtures. 2D PAGE separates soluble proteins with isoelectric points from pI 3-10 and Mw from 10-130 kDa. Proteins spots can be cut out from the gel and identified by a mass spectrometric analysis and database search, providing the protein name and database accession number. Typically 200-500 protein spots can be observed and identified in a silver stained 2D gel.

Workflow

• Sample collection and protein extraction
• Depletion of abundant proteins, such as albumin and immunoglobulins
• 2D electrophoresis with isoelectric focusing (IEF) and molecular weight separation of the proteins
• Protein visualization with quantitative staining methods
• Image analysis – quantitative comparison of gel images and location of differentially expressed proteins
• Spot picking and protein digestion with trypsin
• MALDI-MS/MS peptide analysis
• Database search and protein identification

The 2D image analysis is performed with the Ludesi Redfin software (www.ludesi.com) for optimal spot detection, statistical analysis and reporting.

The identified proteins are reported with online access to the 2D gel images and spot statistics, the quantitative analysis of differential protein spots, and the mass spectrometric protein identification report.