Features of BioID Biotin is a naturally occurring metabolic enzyme cofactor that is active only when covalently linked to the enzyme by the action of a specific protein-biotin ligase. Any biotinylated substrate can bind tightly to protein affin and streptavidin. Its main advantages include: 1. High sensitivity - The affinity between biotin and streptavidin (Kd=10-15 mol/L) is >1,000-fold higher than antibody-mediated interactions, allowing for more efficient and stable capture of protein complexes. 2. High specificity - This approach avoids the use of antibodies, thereby significantly reducing non-specific binding. In addition, the extraordinary stability of biotin-streptavidin allows for rigorous purification to eliminate protein contamination. 3. Highly adaptable - binding of streptavidin to biotin is rapid, specific, and can still occur under conditions where most other proteins have been denatured, such as high temperature or 6M guanidine hydrochloride or 1% sodium dodecyl sulfate (SDS). Biotinylated proteins can be efficiently purified directly from crude extracts by a one-step procedure. In contrast, the most commonly used affinity tags require many purification steps prior to the affinity binding step. BioID Protein Interaction Studies The basic principle of BioID is the fusion expression of biotin ligase and decoy proteins in the target cells. After adding the appropriate amount of biotin, proteins that interact with or are in close proximity to the decoy protein will be labeled with biotin by the biotin ligase. Afterwards, the biotin-labeled proteins are purified using streptavidin and the products are identified by mass spectrometry. The results of the experimental group and the control group will be compared and analyzed to obtain the proteins that interact with the decoy proteins. The core advantage of BioID-MS is that it detects PPIs in the natural cellular environment with essentially no interference with the normal life activities of the cells. The detection range can be modulated by varying the length of the linkage portion between fusion proteins. BioID-MS is also suitable for identifying weak or transient interactions, insoluble protein interactions, and protein interactions mediated by post-translational modifications. It may be more effective than AP-MS in detecting low-abundance proteins. This technique has been used to identify coronavirus replication, HIV virus protein interactions network, bacterial host recognition, mitosis-associated protein interactions, etc. The disadvantage of this method is that it is not possible to determine whether the identified protein is directly or indirectly interacting with the decoy protein. Interacting proteins without a primary amine cannot be biotinylated and are therefore undetectable. Low expression levels of PPI chaperones may also lead to false negatives. In some cases, the biotinylation process itself may have an impact on protein interactions. Creative Proteomics has established a division dedicated to the study of molecular interactions and an advanced proximity-dependent biotin identification (BioID) technology platform to provide solutions for downstream studies following a wide variety of isolations, particularly drug screening, diagnostic assays, protein dynamics, and interaction studies.