2D DIGE saturation labeling for minute sample amounts.
The 2D DIGE technique, based on fluorophores covalently linked to amino acid side chain residues and the concept of an internal standard, has significantly improved reproducibility, sensitivity, and the dynamic range of protein quantification. In saturation DIGE, sulfhydryl groups of cysteines are labeled with cyanine dyes to completion, providing a so far unraveled sensitivity for protein detection and quantification in 2D gel-based proteomic experiments. Only a few micrograms of protein per 2D gel facilitate the analysis of about 2,000 analytes from complex mammalian cell or tissue samples. As a consequence, 2D saturation DIGE is the method of choice when only minute sample amounts are available for quantitative proteome analysis at the level of proteins rather than peptides. Since very low amounts of samples have to be handled in a reproducible manner, saturation DIGE-based proteomic experiments are technically demanding. Moreover, successful saturation DIGE approaches require a strict adherence to adequate reaction conditions at each step. This chapter is dedicated to colleagues already experienced in 2D PAGE protein separation and intends to support the establishment of this ultrasensitive technique in proteomic workgroups. We provide basic guidelines for the experimental design and discuss crucial aspects concerning labeling chemistry, sample preparation, and pitfalls caused by labeling artifacts. A detailed step-by-step protocol comprises all aspects from initial sample preparation to image analysis and statistical evaluation. Furthermore, we describe the generation of preparative saturation DIGE gels necessary for mass spectrometry-based spot identification.
Laboratory for Functional Genome Analysis LAFUGA, Gene Center, Ludwig-Maximilians-University, Munich, Germany. arnold@genzentrum.uni-muenchen.de
Georg J
GJ
Arnold
Thomas
T
Fröhlich
D053000:Analytic Sample Preparation Methods; D005374:Filtration; D006801:Humans; D013058:Mass Spectrometry; D011506:Proteins / Q000032:analysis / Q000737:chemistry / Q000302:isolation & purification; D040901:Proteomics; D013194:Staining and Labeling / Q000379:methods*; D057825:Two-Dimensional Difference Gel Electrophoresis / Q000379:methods*
D011506:Proteins