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

Abstract

AbstractHigh‐throughput western blot (WB) analysis can be used to obtain more consistent, comparable, and informative data from precious samples and materials with extremely limited availability, such as various age‐related, subtype‐specific human induced neurons (hiNs). In this study, p‐toluenesulfonic acid (PTSA), an odorless tissue fixative, was used to inactivate horseradish peroxidase (HRP) and develop a high‐throughput WB method. PTSA‐treated blots demonstrated rapid and efficient HRP inactivation without detectable protein loss or epitope damage. With a brief PTSA treatment (1 min at room temperature [RT]) before every subsequent probing, 10 dopaminergic hiN proteins could be sequentially, sensitively, and specifically detected in the blot. The resulting WB data confirmed the age‐associated and neuron‐specific features of hiNs and revealed a significant reduction in two Parkinson's disease‐associated proteins, UCHL1 and GAP43, in normal aging dopaminergic neurons. Overall, this study developed a unique and high‐efficiency WB analysis method for capturing robust and useful data from limited, precious samples.