A New Method for Quantitative Immunoblotting of Endogenous alpha-Synuclein

作者：Newman Andrew J; Selkoe Dennis; Dettmer Ulf^*

来源：PLos One, 2013, 8(11): e81314.

DOI：10.1371/journal.pone.0081314

beta-Sheet-rich aggregates of alpha-synuclein (alpha Syn) are the hallmark neuropathology of Parkinson's disease and related synucleinopathies, whereas the principal native structure of alpha Syn in healthy cells - unfolded monomer or alpha-helically folded oligomer - is under debate. Our recent crosslinking analysis of alpha Syn in intact cells showed that a large portion of endogenous alpha Syn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying alpha Syn Western blot signals among samples, as crosslinked alpha Syn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased alpha Syn immunoreactivity even after cleavage with the reducing agent beta-mercaptoethanol. The effect was observed with all alpha Syn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous alpha Syn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of alpha Syn after DSP/beta-mercaptoethanol treatment, in contrast to a marked loss of alpha Syn without this treatment. The treatment also enhanced immunodetection of the homologs beta- and gamma-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 alpha Syn to blotting membranes. These data help explain the recent report of fixing alpha Syn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/beta-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance alpha Syn forms such as extracellular and post-translationally modified alpha Syn and splice variants.

• 出版日期2013-11-20