Binding assays were performed as described (28 (link), 40 (link)). Briefly, 96-well plates (Nunc) were preadsorbed with Vn-HX (5 μg/ml), Vn (5 μg/ml; Sigma), or gelatin (20 μg/ml; Sigma), then blocked with tris-buffered saline (TBS) containing 3% milk, and washed with TBS supplemented with 0.05% Tween 20. Recombinant C-terminal His-tagged Ail (Ail-His) was purified, refolded, and concentrated to 80 μg/ml in buffer with 4 mM decylphosphocholine and then added in decreasing concentrations to the preadsorbed wells. After incubating overnight at 4°C, bound Ail-His was detected with mouse anti-His monoclonal antibody (Qiagen), secondary goat anti-mouse antibody conjugated to horseradish peroxidase (Sigma), and the horseradish peroxidase substrate o-phenylenediamine (Pierce).
Protocol detail
Ail Protein Binding Assay
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.
96-well plates
gelatin
mouse anti-His monoclonal antibody
goat anti-mouse antibody conjugated to horseradish peroxidase
Anti antibody
Assays
Buffer
Gelatin
Goat
Horseradish peroxidase
Milk
Monoclonal antibody
Mouse
O phenylenediamine
Saline
Tween 20
Corresponding Organization : Sanford Burnham Prebys Medical Discovery Institute
Other organizations : University of Miami
Top 5 similar protocols
Variable analysis
independent variables
- Concentration of recombinant C-terminal His-tagged Ail (Ail-His)
- Binding of Ail-His to the preadsorbed wells
- Vn-HX (5 μg/ml)
- Vn (5 μg/ml)
- Gelatin (20 μg/ml)
- Tris-buffered saline (TBS) containing 3% milk
- TBS supplemented with 0.05% Tween 20
- Buffer with 4 mM decylphosphocholine
- Vn-HX (5 μg/ml)
- Vn (5 μg/ml)
- Gelatin (20 μg/ml)
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.
Sign up for free or login to display all annotations
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?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!