MiniTurbo Proximity Labeling
MiniTurbo proximity labeling is a biotinylation-based strategy that utilizes an engineered mutant biotin ligase (TurboID) to rapidly and efficiently tag neighboring proteins. Compared to the conventional BioID technique, MiniTurbo significantly enhances labeling speed and efficiency, making it particularly advantageous for dynamic studies and live-cell experiments. MiniTurbo is a genetically modified biotin ligase fused to a target protein to form a fusion protein. Within the cell, when MiniTurbo approaches other proteins, it uses ATP to convert biotin into biotin-AMP. As a reactive intermediate, biotin-AMP covalently tags nearby proteins with biotin. These biotinylated proteins are subsequently enriched using streptavidin-coated beads due to the high specificity and affinity between biotin and streptavidin or avidin, followed by mass spectrometry analysis to identify proteins interacting with or in proximity to the target protein.
Workflow of MiniTurbo Proximity Labeling
1. Genetic Engineering and Plasmid Construction
The first step of MiniTurbo proximity labeling involves constructing an expression vector for the fusion protein. Typically, the TurboID sequence is fused with the target protein gene, and molecular cloning techniques are used to generate the expression plasmid. The plasmid design must ensure that TurboID is efficiently expressed and correctly folded to maintain its biotin ligase activity.
2. Cell Transfection and Stable Cell Line Establishment
The constructed plasmid is introduced into cells via transfection techniques such as electroporation or lipid-based transfection. In some studies, researchers establish stable cell lines expressing the MiniTurbo fusion protein to ensure consistent experimental results.
3. Biotinylation Reaction and Protein Capture
Upon biotin addition in cells, TurboID rapidly biotinylates its neighboring proteins. This process is typically conducted under physiological conditions to preserve biological functions. After cell lysis, streptavidin beads are used to capture the biotinylated proteins.
4. Mass Spectrometry Analysis and Data Interpretation
The captured biotinylated proteins are separated by gel electrophoresis and analyzed by mass spectrometry to identify the proximity proteome. Bioinformatics tools are commonly used to interpret the data and quantify the protein interaction network.
Advantages of MiniTurbo Proximity Labeling
1. Rapid Labeling
MiniTurbo completes biotinylation within 10 minutes, significantly improving experimental efficiency compared to conventional biotin ligase techniques such as BioID, which require several hours for labeling.
2. Low Cytotoxicity
Unlike APEX2, which requires the addition of toxic H₂O₂ for labeling, MiniTurbo does not induce significant cytotoxicity, allowing labeling under conditions that closely mimic physiological states. This is particularly beneficial for studying native protein-protein interactions in cells.
3. High Specificity and Temporal Control
MiniTurbo exhibits minimal background labeling before biotin addition, enabling precise temporal control over protein interaction capture at specific time points or under defined conditions.
4. Detection of Weak and Transient Interactions
Traditional protein interaction detection methods, such as co-immunoprecipitation, are limited to high-affinity interactions. MiniTurbo, however, can detect weak, transient, or hydrophobic interactions under native conditions, facilitating the discovery of previously undetectable protein interactions.
Applications of MiniTurbo Proximity Labeling
1. Protein Interaction Network Analysis
MiniTurbo enables the identification of protein-protein interactions, from single protein interactome mapping to complex interaction network construction, providing insights into cellular signaling pathways.
2. Subcellular Proteomic Mapping
This technique allows for the labeling and identification of proteins within different subcellular structures, aiding in the characterization of protein composition and function in specific cellular compartments.
3. Membrane Contact Site Studies
MiniTurbo facilitates the investigation of protein composition and interactions at membrane contact sites between organelles, contributing to the understanding of intracellular transport and signaling processes.
4. Protein Topology and Surface-Exposed Subunit Identification
It can be used to determine the topology of membrane proteins and identify protein subunits exposed on the cell surface, providing crucial information for functional and mechanistic studies.
5. Protein-Nucleic Acid Interaction Studies
When combined with other techniques, MiniTurbo can identify proteins that associate with specific genomic loci or RNA motifs, offering insights into the roles of protein-nucleic acid interactions in gene regulation.
MtoZ Biolabs offers MiniTurbo proximity labeling services, providing an efficient and precise labeling strategy to support your research needs. Our comprehensive solutions, from plasmid construction to mass spectrometry analysis, ensure reliable data acquisition for your studies. For more information or technical support, please contact us. Our expert team is dedicated to providing professional guidance and customized research solutions to accelerate breakthroughs in your scientific projects.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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