The optimization of protocols for proteome difference gel electrophoresis (DiGE) analysis of preneoplastic skin.
Difference gel electrophoresis (DiGE) allows the reliable comparison of proteome differences between two or three samples within a single gel, by way of a CyDye fluorescent labeling system. This facilitates identification of protein differences avoiding the difficulties associated with gel-to-gel variation. A drawback of this approach is the necessity for high-purity protein samples, since contaminants can interfere with the labeling process, affecting subsequent analysis. Thus far, DiGE has been applied to the study of various sample types derived from relatively simple starting materials such as serum, cell lines, or primary cells. Herein, we describe optimization of protein extraction and purification from a complex tissue (the murine ear) of which a major component is skin, which is compatible with the CyDye labeling system and DiGE. Protein samples obtained by this method from preneoplastic, transgenic tissue have been effectively compared to normal tissue samples to reveal bona fide differences, verifiable by Western blotting. In total, 41 protein differences (21 up- and 20 down-regulated in the pathological samples) were identified by mass spectrometry (MS). This method can therefore form a guide for those wishing to perform DiGE on complex tissues, and is especially useful for samples with relatively insoluble components such as skin.
Hannigan
Adele
A
Division of Molecular Genetics, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G11 6NU, United Kingdom.
Burchmore
Richard
R
Wilson
Joanna B
JB
D000818:Animals; D019295:Computational Biology / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D004817:Epidermis / Q000378:metabolism; D005260:Female; D005456:Fluorescent Dyes / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D013058:Mass Spectrometry; D051379:Mice; D008822:Mice, Transgenic; D011230:Precancerous Conditions / Q000175:diagnosis* / Q000473:pathology; D040901:Proteomics / Q000379:methods*; D012867:Skin / Q000473:pathology*; D012878:Skin Neoplasms / Q000175:diagnosis / Q000473:pathology
D005456:Fluorescent Dyes