96-well plates were coated with purified human integrin αvβ3 (Millipore CC1021, 0.5 μg in 100μl), incubated at 4°C overnight, and blocked with 50 mg/mL BSA for 90 minutes at 30°C. Recombinant human Galectin-3 (R&D Systems 1154-GA-050, 1 μg/well) and test agents were added for a total volume of 50μl, then incubated for 4 hours at 30°C. Wells were washed, fixed with 2% PFA in PBS for 15 minutes at room temperature, washed, and incubated with rat monoclonal Galectin-3 antibody (BioLegend 125401, 0.5 mg/mL diluted 1:100) for 30 minutes on ice. After washing, wells were incubated with secondary antibody (Life Technologies A21210, AF488 rabbit anti-rat IgG diluted 1:200) for 30 minutes on ice. After washing, fluorescence was read using a Tecan Infinite M200 (excitation 485, emission 538) to quantify αvβ3/Galectin-3 binding.
Protocol detail
Quantifying Integrin αvβ3-Galectin-3 Binding
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.
Anti igg
Antibody
Fluorescence
Galectin 3
Human
Human galectin 3
Integrin αvβ3
M200
Monoclonal antibody
Rabbit
Corresponding Organization :
Other organizations : Sanford Consortium for Regenerative Medicine, University of California, San Diego
Top 5 similar protocols
Protocol cited in 3 other protocols
Variable analysis
independent variables
- Concentration of recombinant human Galectin-3 (1 μg/well)
- Concentration and identity of test agents
- Binding of Galectin-3 to immobilized integrin αvβ3, as measured by fluorescence intensity
- Concentration of purified human integrin αvβ3 (0.5 μg in 100μl)
- Blocking with 50 mg/mL BSA for 90 minutes at 30°C
- Incubation time (4 hours at 30°C)
- Primary antibody (rat monoclonal Galectin-3 antibody, 0.5 mg/mL diluted 1:100)
- Secondary antibody (AF488 rabbit anti-rat IgG, diluted 1:200)
- Fluorescence measurement (excitation 485, emission 538)
- Positive control: Binding of Galectin-3 to immobilized integrin αvβ3 in the absence of test agents
- 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.
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!