Cargando…

A critical path to producing high quality, reproducible data from quantitative western blot experiments

Western blotting experiments were initially performed to detect a target protein in a complex biological sample and more recently, to measure relative protein abundance. Chemiluminescence coupled with film-based detection was traditionally the gold standard for western blotting but accurate and repr...

Descripción completa

Detalles Bibliográficos
Autores principales: Taylor, Sean C., Rosselli-Murai, Luciana K., Crobeddu, Bélinda, Plante, Isabelle
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Nature Publishing Group UK 2022
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9585080/
https://www.ncbi.nlm.nih.gov/pubmed/36266411
http://dx.doi.org/10.1038/s41598-022-22294-x
Descripción
Sumario:Western blotting experiments were initially performed to detect a target protein in a complex biological sample and more recently, to measure relative protein abundance. 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. The development of sensitive, camera-based detection technologies coupled with an updated technical approach permits the production of reproducible, quantitative data. 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. 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. The data reveal when and how to apply these detection methods to achieve reproducible data with a stepwise approach to data processing for quantitative analysis.