...discuss the natural role of biotin, biotinylation, the discovery of the biotin-avidin interaction and the...using a biotinylated primer. Biotin can also be enzymatically incorporated during PCR using a biotin labelled...fusion proteins Biotin is an essential vitamin for life Although biotin is most commonly known for its role... of the biotin they need in the small intestine from the microflora that reside there. Biotin is a cofactor...metabolites. Biotin acts as a carboxyl carrier from one compound to the other. These biotin dependent enzymes...entire organism on the biotin carboxyl carrier protein (BCCP). Discovery of the biotin-avidin interaction... supplemented with biotin. Thus something in the egg white was sequestering biotin from being absorbed...

... of neighboring proteins within a cell with biotin. Biotin, is a natural coenzyme, that strongly binds...tagged with biotin using avidin coated purification techniques. Due to the high affinity of biotin for avidin...promiscuous biotin ligase that can efficiently label neighboring proteins The first biotin based proximity...APEX catalyzes the oxidation of biotin-phenol to the short-lived biotin-phenoxyl radical in the presence...improved promiscuous biotin ligase (Kim et al 2016). BioID2 consists of a humanized biotin ligase from A. aeolicus...interacting cell surface proteins with a biotin tagged LPETG peptide. Biotin tagged proteins can then be purified...incubate those cells with exogenous biotin. After a few hours biotinylated proteins can be captured on streptavidin...

...target protein with the vitamin biotin. The interaction between biotin and the egg-white protein avidin...the location of biotin incorporation and to a lesser extent, the number of added biotins per molecule cannot...interaction approaches that of GST-glutathione. Biotin-based immobilization for kinetic analysis Proteolytic...covalent. Therefore, protein immobilization through biotin-avidin binding could provide a true measurement...interactions. Avidin and its derivatives have four biotin-binding sites per molecule so, theoretically, saturated...amine-reactive biotin derivatives that generate covalent bonds with positively-charged amino acids. Unfortunately...selective approach entails the use of the enzyme biotin ligase, which binds to a small (< 20 amino acid...

... with biotin. When proteins Y and Z part ways, BirA* biotinylation activity ceases. After biotin tagging... an E. coli biotin ligase. In BioID, BirA* is fused to a bait protein and it biotinylates proteins that... restored, and nearby proteins are labeled with biotin. For labeling, BirA* fusion proteins are expressed...stable cell lines and these cells are then grown in biotin-containing media. FRET is another example of a ...protein complementation assay where BirA*, an E. coli biotin ligase is split into two non-functional fragments...transient protein-protein interactions since the biotin labeling remains even if the interaction with the..., Roux, K.J., & Raida, M. (2012). A promiscuous biotin ligase fusion protein identifies proximal and interacting...

... labels the proteins in its vicinity with biotin. The biotin-labeled proteins can then be isolated and...amino acid side chains (e.g. Tyrosine) with biotin. These biotin-labeled proteins can them be analyzed by...peroxidase, (APEX) that labels proximal proteins with biotin as an affinity tag. This ‘Caspex’ construct is ...a separate plasmid. When cells are treated with biotin–phenol and H2O2, APEX2 labels electron-rich amino...technology, which they call C-BERST (dCas9–APEX2 biotinylation at genomic elements by restricted spatial tagging...

...choice for ELISAs.  Biotin Biotin is used in a range of antibody-based assays. Biotin itself is not visible...targets. Check out our post on Biotinylation to learn more about biotin and its use in molecular biology...streptavidin, both of which can bind up to four biotin molecules and can themselves be linked to a labeling...allow for efficient signal amplification, making biotin conjugated antibodies good options for detecting...The Basics of Western BlottingPlasmids 101: Biotinylation Resources on Addgene.org Unconjugated antibodies...

...most frequently used with TO1-biotin or TO3-biotin (the addition of biotin allows for the purification ...from cells using this tag, and both TO1-biotin and TO3-biotin contain an acetate modification that reduces...compartmentalisation for their high binding affinity to TO1-biotin. Two of these variants exhibit increased brightness...

...components: dCas9 with an added biotin acceptor site BirA, a biotin ligase a gRNA(s) targeting a single...Original CAPTURE CAPTURE 2.0 Biotinylation enzyme BirA Endogenous biotin ligases Expression system Stable...targeting sequence that is recognized by biotin ligases normally expressed in eukaryotic cells. Using this... Stable cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or more gRNAs Two separate... the biotinalatable tag on dCas9 with a BioTAP-tag. The BioTAP-tag is a 69 aa long biotinylation targeting...the dCas9 to the loci of interest and dCas9 is biotinylated by BirA. From here, the CAPTURE method is similar...enough to be pulled down via streptavidin IP. Biotinylated dCas9 goes along for the ride throughout all...

...cells. BioID was derived from biotin ligase and uses far less toxic biotin for labeling, but the process...applied the same directed evolution technique to the biotin ligase from which BioID was derived, creating a... TurboID and miniTurbo, which utilize non-toxic biotin, but label nearly as quickly as APEX2. What’s more...

...epitope-targeting antibody that is conjugated to a biotin tag but not a fluorophore. In a separate step, ...protein, which binds the primary antibody through the biotin-streptavidin interaction (Figure 1a). This interaction...for panel design. Find streptavidin plasmids and biotin plasmids at Addgene!     Figure 1: Indirect...achieved through two-protein labeling by utilizing(a) biotin-streptavidin binding and (b) antibody host species...protein-tag or protein-protein interactions, like biotin-streptavidin binding and antibody host species ...

...antibody is biotin-conjugated, streptavidin (which has a high affinity for binding biotin), is conjugated...is also conjugated to another molecule, such as biotin. Next, a conjugated substrate is added to the wells...

... To biotinylate the nanobodies, the authors used biotin dissolved in DMSO which allows both biotin and...and biotin-4-fluorescein to be dissolved at a higher concentration, and results in uniform NB biotinylation...pMAL-T-Avi-His/BirA, a vector commonly used for in vivo biotinylation via BirA and AviTAG. pMAK contains the signal...

...of pre-existing transgenic lines, termed BLITZ (Biotin Labeling In Transgenic Zebrafish) (Xiong, et al...Image from Xiong et al. 2021.    BLITZ uses the biotin ligase TurboID fused to a conditionally stabilized...

... rely on the promiscuous E. coli biotin ligase, BirA*, to biotinylate proteins that are within ~10 nm ...detection (RaPID) method. RaPID borrows the E. coli biotin ligase BirA* from BioID and allows a researcher.... BirA* requires 16-18 hours to sufficiently biotinylate interacting proteins, which results in poor spatiotemporal... also tweaked BirA* to increase its rate of biotinylation. The improved enzyme, called BASU, is derived...Affinity Purification (CRAPome), to identify highly biotinylated background proteins reported by other research...

...plasmid-based biotin ligase. Biotinylated dCas9, with bound DNA, can then be purified by streptavidin ...capture (3C) technique. In addition to 3xFLAG and biotin tags, use of tag systems including Protein A, 1xFLAG...dCas9 fused to a biotinylation tag expressed on a plasmid, allowing for in vivo biotinylation through a plasmid-based...2016). 3xFLAG-tag fused to dCas9, along with a biotinylated gRNA, were successfully used in this "in vitro...In situ capture of chromatin interactions by biotinylated dCas9." Cell 170.5 (2017): 1028-1043. PubMed...

...drastically reducing background labeling caused by the biotin naturally present in experimental systems. They... group for click chemistry reactions instead of biotin — a valuable development in and of itself. After...illuminated with blue light, catalyzing the biotinylation of nearby proteins. B) TransitID uses sequential...

...Immunohistochemical Staining Technique Adaptable to Biotin-Free Reagents, Immunofluorescence, and Multiple...

...version of the promiscuous biotin ligase used in their popular biotinylation method for identifying protein-protein...method uses a bait protein fused to a biotin ligase to biotinylate proximate proteins in the cell. These...These candidate proteins can be enriched via biotin pull-down methods and identified using mass spectrometry...selective targeting of fusion proteins, requires less biotin, and shows enhanced labeling of proximate proteins...Acids Res. 2016. PubMed PMID: 26969735 Updated biotinylation tools to identify proximate proteins The Roux...

... Engineered ligases (e.g., lipoic acid ligase, biotin ligase, phosphopantetheinyltransferase) – These...

...molecular biology studies. For example the E. coli biotin ligase, BirA, has been used to identify and study...