Auto-(de)phosphorylation assay

This protocol describes an assay to measure ATP13A4 phospho-enzyme levels. ATP13A4, a P-type ATPase, forms a phosphorylated intermediate on a conserved aspartate residue during its catalytic cycle. In P5B ATPases like ATP13A4, spontaneous auto-phosphorylation occurs, followed by polyamine-induced dephosphorylation. The assay utilizes SDS-PAGE under acidic conditions, preserving the stability of the aspartyl-phosphate intermediate, followed by autoradiography to detect [32P]-labeled phosphorylated intermediates.