Isolation and Imaging of His- and RFP-tagged Amyloid-like Proteins from by TEM and SIM.

In our recently published paper, we highlight that during normal aging of age-dependent aggregates of proteins form and lead to functional decline of tissues. The protocol described here details the isolation of two proteins from in their aggregated amyloid-like form, casein kinase I isoform alpha (KIN-19) and Ras-like GTP-binding protein rhoA (RHO-1). We used nickel beads to isolate His-tagged KIN-19 and RHO-1, and thus permitting the isolation of both small and large aggregated or fibrillary forms of the proteins. We characterized their morphology by transmission electron microscopy. We further expressed RFP-tagged proteins and stained them with a fluorescent molecule, thioflavin T, which identifies β-sheet structures, and which is a defining feature of amyloid fibrils. We further applied structured illumination microscopy to determine the level of colocalization between RFP and thioflavin T.