Single-molecule ACTR-NCBD binding assay

The single-molecule binding experiments were conducted as described before (7 (link)). In short, adhesive silicone hybridization chambers (Secure Seal Hybridization Chambers, SA8R-2.5; Grace Bio-Labs) were fixed onto PEGylated, biotinylated quartz coverslips (Bio_01; MicroSurfaces, Inc.); 0.2 mg/mL Avidin D (Vector Labs) were incubated for 5 min in a reaction chamber, followed by addition of 10 pM ACTR–Cy3B to yield a surface coverage of 0.1 to 0.3 molecules per µm². Binding experiments were conducted in the appropriate PEG solution, supplied with 16 nM CF680R-labeled NCBD, 0.01% Tween 20, 1% (wt/vol) glucose, 0.4 mg/mL glucose oxidase, 400 U/mL catalase as oxygen scavenging system, as well as 1 mM methyl viologen and 1 mM ascorbic acid as triplet quenchers. Addition of these components led to a 0.1-fold dilution of the PEG solution, which was taken into account.

Partial Protocol Preview
This section provides a glimpse into the protocol.
The remaining content is hidden due to licensing restrictions, but the full text is available at the following link: Access Free Full Text.

Zosel F., Soranno A., Buholzer K.J., Nettels D, & Schuler B. (2020). Depletion interactions modulate the binding between disordered proteins in crowded environments. Proceedings of the National Academy of Sciences of the United States of America, 117(24), 13480-13489.

Publication 2020

Avidin D

Ascorbic acid Avidin Catalase Dilution Glucose Glucose oxidase Hybridization Methyl viologen Oxygen Quartz Seal Silicone Triplet Tween 20 Vector

Corresponding Organization :

Other organizations : University of Zurich, Washington University in St. Louis

Top 5 similar protocols

Variable analysis

independent variables

Concentration of ACTR-Cy3B (10 pM)
Concentration of CF680R-labeled NCBD (16 nM)

dependent variables

Surface coverage of ACTR-Cy3B (0.1 to 0.3 molecules per μm²)

control variables

Incubation time of Avidin D (5 min)
Concentration of Avidin D (0.2 mg/mL)
Composition of the PEG solution (with 0.01% Tween 20, 1% (wt/vol) glucose, 0.4 mg/mL glucose oxidase, 400 U/mL catalase, 1 mM methyl viologen, and 1 mM ascorbic acid)

controls

Positive control: Not explicitly mentioned.
Negative control: Not explicitly mentioned.

Annotations

Based on most similar protocols

Etiam vel ipsum. Morbi facilisis vestibulum nisl. Praesent cursus laoreet felis. Integer adipiscing pretium orci. Nulla facilisi. Quisque posuere bibendum purus. Nulla quam mauris, cursus eget, convallis ac, molestie non, enim. Aliquam congue. Quisque sagittis nonummy sapien. Proin molestie sem vitae urna. Maecenas lorem.

As authors may omit details in methods from publication, our AI will look for missing critical information across the 5 most similar protocols.

About PubCompare

Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.

We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.

However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.

Ready to get started?

Revolutionizing how scientists
search and build protocols!