A New Method for Quantitative Immunoblotting of Endogenous a-Synuclein

b-Sheet-rich aggregates of a-synuclein (aSyn) are the hallmark neuropathology of Parkinson’s disease and related synucleinopathies, whereas the principal native structure of aSyn in healthy cells- unfolded monomer or a-helically folded oligomer- is under debate. Our recent crosslinking analysis of aSyn in intact cells showed that a large portion of endogenous aSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying aSyn Western blot signals among samples, as crosslinked aSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased aSyn immunoreactivity even after cleavage with the reducing agent b-mercaptoethanol. The effect was observed with all aSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous aSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of aSyn after DSP/b-mercaptoethanol treatment, in contrast to a marked loss of aSyn without this treatment. The treatment also enhanced immunodetection of the homologs b- and c-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of aSyn to blotting membranes. These data help explain the recent report of fixin