title: Application of multiplexed cysteine-labeled complex protein sample for 2D electrophoretic gel alignment.
abstract: The analysis of cellular subproteomes by 2DE is hampered by the difficulty of aligning gel images from samples that have very different protein composition. Here, we present a sensitive and cost-effective fluorescent labeling method for analyzing protein samples that is not dependent on their composition. The alignment is guided by inclusion of a complex mixture of proteins that is co-run with the sample. Maleimide-conjugated fluorescent dyes Dy-560 and Dy-635 are used to label the cysteine residues of the sample of interest and the alignment standard, respectively. The two differently labeled mixtures are then combined and separated on a 2D gel and, after selective fluorescence detection, an unsupervised image registration process is used to align the protein patters. In a pilot study, this protocol significantly improved the accuracy of alignment of nuclear proteins with total cellular proteins.
authors: Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Babtai LT, Kaunas District, Lithuania.
authors: Perttu
authors: P
authors: Haimi
authors: Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Babtai LT, Kaunas District, Lithuania.
authors: Sidona
authors: S
authors: Sikorskaite-Gudziuniene
authors: Institute of Horticulture, Lithuanian Research Centre for Agriculture and Forestry, Babtai LT, Kaunas District, Lithuania.
authors: Danas
authors: D
authors: Baniulis
mesh_terms: D001578:Benzopyrans / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D007091:Image Processing, Computer-Assisted; D007211:Indoles / Q000737:chemistry; D008301:Maleimides / Q000737:chemistry; D027845:Malus / Q000737:chemistry; D010865:Pilot Projects; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry; D012984:Software
chemical_list: D001578:Benzopyrans; C439045:DY 635; D005456:Fluorescent Dyes; D007211:Indoles; D008301:Maleimides; D010940:Plant Proteins; C043592:maleimide; D003545:Cysteine
keywords: Electrophoresis; Fluorescent labeling; Image registration; Subproteomes; Technology