A simple, flexible and high-efficiency western blot analysis for age-related human induced neurons

Abstract

ABSTRACTHigh-throughput western blot (WB) analysis of small and precious samples, such as various age-related subtype-specific human induced neurons (hiNs), confers the ability to obtain more consistent, comparable, and informative data from materials with extremely limited availability. In this study, p-toluenesulphonic acid (PTSA), an odorless tissue fixative, was used to inactivate HRP for developing a high-throughput WB method. PTSA-treated blots showed fast and efficient inactivation of HRP without detectable protein loss and epitope damage. With a brief PTSA-treatment before every next probing, 10 proteins of dopaminergic hiNs could be sequentially, sensitively, and specifically detected in a blot. These WB data proved the age-associated and neuron-specific features of hiNs and further revealed a sharp reduction of two Parkinson’s disease-associated proteins, UCHL1 and GAP43, in the normal aging dopaminergic neurons. Together, this study developed a unique and high-efficiency WB analysis and pinpointed its special value for capturing robust useful data from limited, precious samples.GRAPHICAL ABSTRACTHIGHLIGHTS✓P-toluenesulphonic acid (PTSA) quickly and fully deactivated HRP on immunoblots.✓PTSA was an odorless, non-volatile, low cost, and user-friendly HRP inactivator.✓PTSA allowed high-efficiency WB analysis to save small precious samples and time.✓10 proteins were detected in a single blot of age-relevant human induced neurons.✓UCHL1 and GAP43 sharp decline occurred in aging human induced dopaminergic neurons.