Serial isoelectric focusing as an effective and economic way to obtain maximal resolution and high-throughput in 2D-based comparative proteomics of scarce samples: proof-of-principle.
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SourceJournal of Proteome Research, 4, 6, (2005), pp. 2364-2368
Article / Letter to editor
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Laboratory of Genetic, Endocrine and Metabolic Diseases
Journal of Proteome Research
SubjectDCN 1: Perception and Action; DCN 2: Functional Neurogenomics; DCN 3: Neuroinformatics; IGMD 3: Genomic disorders and inherited multi-system disorders; IGMD 4: Glycostation disorders; IGMD 8: Mitochondrial medicine; NCEBP 10: Human Movement & Fatigue; NCMLS 4: Energy and redox metabolism; ONCOL 3: Translational research; UMCN 3.1: Neuromuscular development and genetic disorders; UMCN 5.3: Cellular energy metabolism; NCEBP 10: Human Movement & Fatigue
In 2D-based comparative proteomics of scarce samples, such as limited patient material, established methods for prefractionation and subsequent use of different narrow range IPG strips to increase overall resolution are difficult to apply. Also, a high number of samples, a prerequisite for drawing meaningful conclusions when pathological and control samples are considered, will increase the associated amount of work almost exponentially. Here, we introduce a novel, effective, and economic method designed to obtain maximum 2D resolution while maintaining the high throughput necessary to perform large-scale comparative proteomics studies. The method is based on connecting different IPG strips serially head-to-tail so that a complete line of different IPG strips with sequential pH regions can be focused in the same experiment. We show that when 3 IPG strips (covering together the pH range of 3-11) are connected head-to-tail an optimal resolution is achieved along the whole pH range. Sample consumption, time required, and associated costs are reduced by almost 70%, and the workload is reduced significantly.
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