BIoID2, short for Biotin-based Identification of Interacting Proteins 2, is an innovative proximity-dependent proteomics technology developed to study protein-protein interactions and spatial proteomics. It is an improved version of its predecessor, BioID, and offers enhanced accuracy and reduced background noise.
The fundamental principle behind BIoID2 involves fusing a bait protein of interest with a modified form of the biotin ligase enzyme called BirA*. When the bait protein comes into proximity with interacting proteins in the cellular environment, BirA* biotinylates the nearby proteins by covalently attaching biotin molecules to specific lysine residues. These biotinylated proteins can then be isolated and identified using affinity purification and mass spectrometry-based proteomic analysis.
By employing BIoID2, you can gain valuable insights into protein-protein interactions, protein complex composition, protein localization, and functional proteomics. This technology enables the systematic mapping of protein interactions, the identification of protein complexes, and the investigation of protein localization within cells.
A truncation fragment of BioID2 with promiscuous biotinylation activity (Zhao et al., 2021).
Design and Construction of Bait Protein: A bait protein that represents the protein of interest is selected and genetically fused with BirA*. The resulting fusion protein is expressed in the target cell line or organism.
Biotin Labeling: Upon expression, the bait protein-BirA* fusion protein is introduced into the cellular environment. In the presence of biotin and ATP, BirA* catalyzes the covalent attachment of biotin to proximal lysine residues on interacting proteins.
Cell Lysis: To access the biotinylated proteins, the cells expressing the bait protein-BirA* fusion are lysed, resulting in the release of cellular contents.
Isolation of Biotinylated Proteins: The biotinylated proteins are selectively enriched using streptavidin-coated magnetic beads or columns. Streptavidin has a high affinity for biotin, allowing for efficient capture and isolation of the biotinylated proteins from the complex protein mixture.
Proteomic Analysis: The isolated biotinylated proteins are subjected to proteomic analysis, typically involving mass spectrometry. This analysis enables the identification and quantification of the interacting proteins.
Data Analysis and Interpretation: The generated data is processed, and bioinformatics tools are employed to analyze and interpret the results. This step aids in identifying protein-protein interactions and unraveling the functional implications of the bait protein.
Creative Proteomics offers comprehensive BIoID2 services to support your understanding of protein interactions and their functional implications. Our team of experienced scientists utilizes state-of-the-art equipment and cutting-edge methodologies to ensure accurate and reliable results.
Our BIoID2 services include:
Experimental Design and Consultation: Our experts collaborate closely with researchers to design BIoID2 experiments tailored to their specific research goals. We provide valuable insights and guidance throughout the experimental planning process.
Bait Protein Design and Construction: Our team assists in the design and construction of bait proteins fused with BirA*. We employ advanced molecular biology techniques to generate high-quality fusion constructs.
Experimental Execution and Optimization: We perform BIoID2 experiments in a controlled laboratory environment, optimizing the experimental conditions to maximize the accuracy and sensitivity of the technique.
Proteomic Analysis and Data Interpretation: We employ cutting-edge mass spectrometry-based proteomic techniques to analyze the biotinylated proteins. Our bioinformatics experts process and interpret the data, providing comprehensive reports and insights into the identified protein interactions.
Customized Solutions: We offer customized BIoID2 solutions to meet specific research requirements, ensuring that our services align with the individual needs of our clients.
Mapping Protein-Protein Interactions: BIoID2 enables the systematic mapping of protein-protein interactions in various cellular contexts. By identifying the interacting partners of a bait protein, researchers can uncover new protein networks and elucidate the underlying mechanisms of cellular processes.
Identifying Protein Complexes: BIoID2 can identify protein complexes by identifying proteins that are in close proximity to the bait protein. This information aids in understanding the composition and dynamics of protein complexes involved in various cellular processes.
Investigating Protein Localization: BIoID2 can provide insights into the spatial distribution of proteins within cells. By selectively biotinylating proximal proteins, BIoID2 allows for the identification of proteins localized in specific subcellular compartments.
Functional Proteomics: BIoID2 facilitates functional proteomics studies by linking bait proteins to their interacting partners. This approach aids in deciphering the roles of individual proteins within cellular pathways and processes.