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

High-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. Graphical and Lay Summary Inactivation of horseradish peroxidase (HRP) facilitates multiple reprobing of a single blot to sequentially detect different proteins instead of rerunning several gels and blots. In this study, the authors demonstrate that p-toluenesulfonic acid (PTSA) inactivates HRP rapidly and efficiently, allowing simple, flexible, and high-efficiency western blot (WB) analysis of diverse proteins on a single blot for age-related, subtype-specific human induced neurons. The results imply the promising potential of PTSA as an excellent and user-friendly HRP-inactivating reagent for novel high-throughput WB analysis, with considerable importance for research on limited, precious samples.