Optimizing the difference gel electrophoresis (DIGE) technology.
Difference gel electrophoresis (DIGE) technology has been used to provide a powerful quantitative component to proteomics experiments involving 2D gel electrophoresis. DIGE combines spectrally resolvable fluorescent dyes (Cy2, Cy3, and Cy5) with sample multiplexing for low technical variation, and uses an internal standard methodology to analyze replicate samples from multiple experimental conditions with unsurpassed statistical confidence for 2D gel-based differential display proteomics. DIGE experiments can facilely accommodate sufficient independent (biological) replicate samples to control for the large interpersonal variation expected from clinical samples. The use of multivariate statistical analyses can then be used to assess the global variation in a complex set of independent samples, filtering out the noise from technical variation and normal biological variation thereby focusing on the underlying variation that can describe different disease states. This chapter focuses on the design and implementation of the DIGE methodology employing the use of a pooled-sample internal standard in conjunction with the minimal CyDye chemistry. Notes are also provided for the use of the alternative saturation labeling chemistry.
Friedman
David B
DB
Proteomics Laboratory, Mass Spectrometry Research Center, Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA.
Lilley
Kathryn S
KS
D000465:Algorithms; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D005456:Fluorescent Dyes; D006801:Humans; D015999:Multivariate Analysis; D020543:Proteome / Q000302:isolation & purification*; D040901:Proteomics / Q000379:methods* / Q000592:standards / Q000706:statistics & numerical data; D012015:Reference Standards; D012680:Sensitivity and Specificity; D012984:Software; D013194:Staining and Labeling
D005456:Fluorescent Dyes; D020543:Proteome