Immunoscreening of fusion proteins produced in lambda plaques
1Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
Current Protocols in Molecular Biology
|February 12, 2008
Summary
This study presents antibody-based screening for identifying protein-expressing plaques, offering an alternative to DNA probes. An enhanced method boosts recombinant protein yield by inducing lac promoter expression.
Area of Science:
- Molecular Biology
- Biochemistry
- Immunology
Background:
- Screening for specific proteins within large libraries of bacteriophage plaques is crucial for molecular biology research.
- Traditional methods often rely on radioactive DNA probes, which can have limitations in terms of safety and specificity for protein targets.
Purpose of the Study:
- To describe a method for screening bacteriophage plaques using antibodies specific to target proteins.
- To provide an alternative protocol for increasing recombinant protein production in plaques.
Main Methods:
- Utilizing antibodies specific to desired proteins for plaque screening.
- Employing induction of the lac promoter to enhance recombinant protein expression within plaques.
Main Results:
- Demonstrated successful screening of plaques using antibody-based methods.
- Showcased an effective strategy to amplify recombinant protein levels per plaque.
Conclusions:
- Antibody-based screening offers a viable alternative to DNA probe screening for identifying protein-containing plaques.
- Inducible expression systems can significantly increase the yield of target recombinant proteins in plaque-based assays.
Related Concept Videos
01:24Tagging and Fusion Proteins
Proteins are involved in several cellular processes and biochemical reactions. Analyzing a specific protein of interest requires it to be isolated from the other proteins in the cell. This is achieved by overexpressing the specific gene in a suitable host to produce large quantities of the target protein. A tag or label is recombined with the gene to produce a fusion protein containing the target protein and the tag. The tags on these fusion proteins can then be used for easy detection and...
01:31Hybridoma Technology
Hybridoma technology is used for the large-scale production of monoclonal antibodies. Monoclonal antibodies bind to only a single antigenic determinant or epitope. Such antibodies are used in research, diagnostics, and disease therapy. The hybridoma technology established in 1975 by Georges Köhler and Cesar Milstein was awarded the Nobel Prize in Medicine in 1984 for revolutionizing research and therapy.
Hybridoma Selection
Commonly used fusion techniques — electroporation, polyethylene glycol...
Hybridoma Selection
Commonly used fusion techniques — electroporation, polyethylene glycol...