CR stain was used as an amyloid detection assay to confirm the amyloid features for amyloid-like proteins. DOPA-containing proteins can be specifically stained by nitro blue tetrazolium (NBT) in glycinate solutions because they can catalyze redox-cycling reactions at an alkaline pH (29). The NBT assay was thus used to confirm the successful tyrosinase modification of TLC-M.

Sixty microliters of protein samples (50 μg/ml) were spotted onto Protran BA83 nitrocellulose membranes (Whatman) with a dot-blot manifold (Schleicher & Schuell Minifold-I Dot-Blot System). For the CR stain assay, the membranes spotted with proteins were immersed in 20 ml of 0.0025 (m/v %) CR solution at room temperature for 1 hour, then taken out and washed three times with copious amounts of deionized water, and incubated in deionized water overnight. For the NBT stain assay, the membranes spotted with proteins were incubated in 20 ml of freshly made NBT solution (0.6 mg/ml) in 2 M potassium glycinate buffer (pH 10.0) at room temperature in a dark place (covered with aluminum foil) for 30 min. The membranes were washed twice with 10 ml of 0.16 M sodium borate solution and soaked in another 20 ml of sodium borate solution overnight. Optical images of the stained samples were captured with a camera. Data are presented in fig. S1 (A and C).

To further characterize the amyloid features of the amyloid nanofiber coatings, we used polarized light microscopy to confirm the green birefringence feature of CR-stained samples. Specifically, 20 μl of dialyzed protein solution (2 mg/ml) was pipetted onto a glass coverslip. After incubation overnight at 4°C, the samples were dried at room temperature, followed by immersion into 1 ml of 0.0025 (m/v %) CR solution at room temperature for 1 hour and washed 10 times with deionized water to thoroughly remove the nonspecifically bound CR dye. Last, the CR-stained samples were observed under an optical microscopy equipped with crossed polarizers (Leica M205 FA). Data are presented in fig. S1B.

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。