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Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions
Published on: August 2, 2015
Investigating protein-protein interactions by far-Westerns
Catherine S Chan1, Tara M L Winstone, Raymond J Turner
1Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, T2N 1N4, Calgary, AB, Canada.
Advances in Biochemical Engineering/Biotechnology
|January 26, 2008
Summary
Far-Western blotting is a common technique to identify protein interaction partners, aiding in protein function elucidation. This guide details its variations, applications, and troubleshooting for researchers.
Area of Science:
- Biochemistry
- Molecular Biology
- Proteomics
Background:
- Protein interactions are crucial for cellular functions.
- Identifying interaction partners helps infer protein function via the 'guilty by association' principle.
- Functional assays for protein binding are essential when direct methods are unavailable.
Purpose of the Study:
- To provide a comprehensive overview of far-Western blotting techniques.
- To guide researchers in selecting appropriate far-Western variations based on experimental questions and available resources.
- To highlight the versatility and practical application of far-Westerns through unique case studies.
Main Methods:
- Detailed discussion of the mechanics and variations of far-Western blotting.
- Inclusion of practical examples and troubleshooting guidance.
- Focus on simplicity and clarity of results inherent to the technique.
Main Results:
- Far-Western blotting remains a powerful and commonly used method in laboratory settings.
- The technique offers simplicity and clarity in analyzing protein-protein interactions.
- Various adaptations of the method cater to diverse research questions.
Conclusions:
- Far-Western blotting is a valuable and versatile tool for identifying protein interaction partners.
- Understanding its variations and troubleshooting is key to successful application.
- The method aids in functional characterization of proteins and serves as a functional assay.
Related Concept Videos
01:15Western Blotting
Western blotting is an analytical technique for protein identification. It has various applications in immunology and medicine, including detecting diseases like bovine spongiform encephalopathy, mad cow disease, and human and feline immunodeficiency virus from biological samples.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
The technique begins with separating proteins from the sample using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by protein transfer, immunoblotting, and finally, protein detection.
02:26Protein Networks
An organism can have thousands of different proteins, and these proteins must cooperate to ensure the health of an organism. Proteins bind to other proteins and form complexes to carry out their functions. Many proteins interact with multiple other proteins creating a complex network of protein interactions.
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein,...
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein,...
02:04Protein-protein Interfaces
Many proteins form complexes to carry out their functions, making protein-protein interactions (PPIs) essential for an organism's survival. Most PPIs are stabilized by numerous weak noncovalent chemical forces. The physical shape of the interfaces determines the way two proteins interact. Many globular proteins have closely-matching shapes on their surfaces, which form a large number of weak bonds. Additionally, many PPIs occur between two helices or between a surface cleft and a polypeptide...
01:19Protein Dynamics in Living Cells
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...