A reliable western blot workflow with improved dynamic range for the detection of myelin proteins in murine brain.

Abstract

Myelin is a highly structured multilamellar sheath produced by oligodendrocytes, which insulates neuronal axons to facilitate neurotransmission. Maturation of oligodendrocytes in cortical regions of the developing murine brain occurs postnatally and corresponds to the marked upregulation of myelin-specific genes. Western blotting is a conventional technique used to study protein expression but historically has only been considered semi-quantitative. This study aims to optimize a western blot workflow for quantification of myelin proteins in murine brain including the examination of the following parameters: sample preparation, electrophoretic transfer conditions, detection parameters, data normalization and linear dynamic range. As a proof of principle, the optimized protocol was employed to characterize both the absolute and relative expression of myelin oligodendrocyte glycoprotein (MOG) throughout neurodevelopment. A dynamic loading paradigm, which varied total protein load across different age groups to ensure antigen detection remained in the linear dynamic range of the assay, showed a greater relative increase in expression when compared to standard loading paradigm. This approach resulted in comparable MOG expression profiles from both absolute and relative quantification. The optimized western blot workflow will facilitate protein quantification and will improve data reproducibility when investigating the molecular mechanisms of myelination in development, aging and disease.