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

Abstract

High-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 Parkinsons 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 ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=143 SRC="FIGDIR/small/526150v2_ufig1.gif" ALT="Figure 1"> View larger version (25K): org.highwire.dtl.DTLVardef@18fcd9dorg.highwire.dtl.DTLVardef@42426org.highwire.dtl.DTLVardef@1e13accorg.highwire.dtl.DTLVardef@1db2f39_HPS_FORMAT_FIGEXP M_FIG C_FIG HIGHLIGHTS{checkmark} P-toluenesulphonic acid (PTSA) quickly and fully deactivated HRP on immunoblots. {checkmark}PTSA was an odorless, non-volatile, low cost, and user-friendly HRP inactivator. {checkmark}PTSA allowed high-efficiency WB analysis to save small precious samples and time. {checkmark}10 proteins were detected in a single blot of age-relevant human induced neurons. {checkmark}UCHL1 and GAP43 sharp decline occurred in aging human induced dopaminergic neurons.