Fig. 1: Optimized pre-cleavage FUS (PC-FUS) protocol.
From: FUS nanoclusters are a distinct state within the dilute phase
A The schematic diagram of the structure of Maltose Binding Protein (MBP)-tagged FUS. The order of the motifs, starting from the N-terminus, is 6xHis tag, MBP-tag, Tobacco Etch Virus (TEV)-site and FUS. B The schematic diagram demonstrates the lagging time of TEV cleavage. C A titration of TEV concentration of 2.5, 12.5, 25 and 50 U/mL, incubating for 5 minutes (lane 2, 4, 6, 8) and 10 minutes (lane 3, 5, 7, 9). D Time course of TEV cleavage. 2.5 U/mL of TEV was added, and an aliquot of reaction was quenched at certain time points from 5 minutes to 1 hour after the start of reaction. E Major steps of pre-cleavage protocol. Step 1, purify the 6xHis-MBP-FUS using a Ni-column. Step 2, add TEV to the product from step 1 under high urea and KCl condition, and incubate for 8 hours. Step 3, apply the mixture to a Ni-column, and collect the flow-through, which is the pre-cleaved FUS. Step 4, apply high imidazole concentration to elute the bound portion for SDS-PAGE. F SDS-PAGE for the major steps of pre-cleaved FUS preparation. Lane 1-4 represents step 1–4 mentioned in (E). Protein preparation and purity were consistent across >5 independent preparations, with similar results obtained each time.
