In Fig. 2, samples were folded and PEG-purified, and the MgCl2 concentration was adjusted to 30 mM. After UV irradiation, the buffer was exchanged to the target buffer/solvent by using ultracentrifugation. Before gel electrophoresis, the samples were incubated for around 2 to 3 hours at room temperature. Samples for the temperature screen were incubated for 30 min at the indicated temperatures. The samples for negative-stain TEM were prepared at a monomer concentration of 50 nM, with incubation on the grid for 3 to 5 min.

In Fig. 3B, the stability screen in folding buffer (5 mM MgCl2) supplemented with 10 % FBS (not heat-inactivated; Gibco; A3160801, Thermo Fisher Scientific) was performed at a monomer concentration of 20 nM at 37°C for the indicated time. The samples were frozen in liquid nitrogen and analyzed using agarose gel electrophoreses. In Fig. 3C, all nucleases were purchased from New England Biolabs and used at a concentration of 100 U/ml in the supplied manufacturer’s buffer. The samples (10 nM) were incubated at 37°C for 24 hours. In Fig. 3D, the time course of the stability against DNase I nuclease digestion was performed at a monomer concentration of 10 nM in the supplied DNase I buffer at 37°C.

In Fig. 5, the irradiation time screen for the switch was performed in triplicate. The irradiated volume was 25 μl at a monomer concentration of 5 nM. For the analysis of the gel shown in Fig. 5B, we calculated the ratio between the band including closed particles and the bands including open and closed particles. The grayscale values for each band were obtained by integration. The data points in Fig. 5C represent the average, and the error bars represent the SD of the three independent experiments. For the assembly of the filaments, monomers were folded and PEG-purified. The pellet was dissolved in folding buffer (5 mM MgCl2) to obtain a monomer concentration of 100 nM. After equilibration, the MgCl2 concentration was adjusted to 20 mM, and the sample was incubated at 40°C for 3 days in the Tetrad to obtain filaments. One part of the sample was irradiated at 310 nm for 30 min. The MgCl2 concentration was decreased to 5 mM by the addition of EDTA.

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.