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A methodology to optimize and produce excellent, quantitative western blot results with rigorous data analysis from membranes probed with both fluorescent and chemiluminescent antibodies is described. Fluorescence reagent and detection solutions are the latest innovation in western blotting but there remains questions and debate concerning their relative sensitivity and dynamic range versus chemiluminescence. The development of sensitive, camera-based detection technologies coupled with an updated technical approach permits the production of reproducible, quantitative data. Chemiluminescence coupled with film-based detection was traditionally the gold standard for western blotting but accurate and reproducible quantification has been a major challenge from this methodology. Western blotting experiments were initially performed to detect a target protein in a complex biological sample and more recently, to measure relative protein abundance. |
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