To produce LLPS-induced coatings on flat substrates, the inside wall of microtubule or three-dimensional (3D) microfluidic device, DOPA-modified TLC-M solution was first concentrated to 2 mg/ml after purification and then dialyzed against the dialysate buffers [20 mM tris-HCl, 50 mM NaCl, and 0.5 mM EDTA (pH 5)] at 4°C for 1 hour to remove excess NaCl and imidazole and produce solution containing high density of liquid-like droplets. Then, we added 2 ml of dialyzed DOPA-modified TLC-M protein solutions (2 mg/ml) to entirely cover the surface of cut flat substrates inside each well of a 24-well plate or injected dialyzed DOPA-modified TLC-M protein solutions (2 mg/ml) into a microtubule. The samples were then incubated overnight at 4°C. A thin layer of nanofiber coatings could form on the substrates or the inside wall of microtubule. Data are presented in Fig. 4 (A to C) and fig. S8.

Microfluidic devices made of polydimethylsiloxane (PDMS) were fabricated in J.Z.’s lab at the State Key Laboratory of Supramolecular Structure and Materials of Jilin University. To prepare protein coatings inside a microfluidic device, a PDMS microfluidic device was compressed onto a glass slide surface and connected to a syringe (1 ml) using a Teflon pipeline. DOPA-modified TLC-M liquid-like droplet solution was slowly injected into the microfluidic device channel. After incubation overnight at 4°C, copious amounts of deionized water were applied to wash the microfluidic device channel to remove any loosely bound proteins. Data are presented in Fig. 4D.

To produce LLPS-induced coatings in buffers with different pH values or different ionic strengths, DOPA-modified TLC-M solution was first concentrated to 2 mg/ml after purification and then dialyzed against the dialysate buffers (20 mM tris-HCl, 50 mM NaCl, and 0.5 mM EDTA) with different pH values (3, 5, 7, 9, and 11) or dialysate buffers [20 mM tris-HCl and 0.5 mM EDTA (pH 5)] with different NaCl concentrations (0.05, 0.2, 0.5, and 1.0 M) at 4°C for 1 hour. Then, we placed the cut flat substrates inside a 24-well plate. Two milliliters of dialyzed DOPA-modified TLC-M protein solutions were then added to entirely cover the surface of substrates. The samples were then incubated overnight at 4°C. A thin layer of nanofiber coatings could form on the substrates. Data are presented in Fig. 2 (E to G).

注意:以上内容是从某篇研究文章中自动提取的,可能无法正确显示。



Q&A
请登录并在线提交您的问题
您的问题将发布在Bio-101网站上。我们会将您的问题发送给本研究方案的作者和具有相关研究经验的Bio-protocol成员。我们将通过您的Bio-protocol帐户绑定邮箱进行消息通知。