Isolation and Purification of Amyloid Fibers

The modified method of amyloid fibers isolation were based on two methods described by Schwartz et al. (2012 (link)) and Romero et al. (2010 (link)). biofilms were grown in eight 10 cm² dishes for 24 h. After washing by PBS for two times, biofilms were scraped and diluted in 1 ml/dish saline extraction buffer (5 mM potassium phosphate, 2 mM MgCl2, 100 mM morpholi nepropane sulphonic acid (Mops) and 1 M NaCl, PH = 7) supplemented with a protease inhibitor mixture. The biofilm suspensions were homogenized using a tissue homogenizer (TissueMiser, Fisher) to shear fibers free from the cell walls. Supernatants were clarified by repeated centrifugation (seven times) at 7000 rpm for 2 min to remove bacteria. At last, supernatants without cells were centrifugation at 16,000g for 20 min to precipitate amyloid fibers, and the precipitate was redissolved in distilled deionized water. Presence of fibers was confirmed via TEM imaging. Amyloid fibers solution was treated by DNase I, RNase and protease K. After treating, amyloid fibers were confirmed by TEM.