Enzymatic Biotinylation
Enzymatic Biotinylation - Related Videos
09:13
Cell-surface Biotinylation Assay
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2023
A cell can regulate the amount of particular proteins on its cell membrane through endocytosis, following which cell surface proteins are effectively sequestered in the cytoplasm. Once within a cell, these surface proteins can be either destroyed or “recycled” back to the membrane. The cell surface biotinylation assay provides researchers with a way to study these phenomena. The technique makes use of a derivative of the small molecule biotin, which can label surface proteins and then be...
11:32
Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen
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Cited by 20 •
2017
Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.
06:43
In Vivo Proximity Biotinylation for Protein Interaction Studies in Paramecium tetraurelia
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Cited by 1 •
2025
The protocol presents a method for in vivo covalent attachment of biotin to proteins based on their proximity to a biotin ligase fused to a protein of interest. This modification allows for a selective enrichment of the proteins using streptavidin beads as needed in protein interaction studies.
12:03
Purification of Biotinylated Cell Surface Proteins from Rhipicephalus microplus Epithelial Gut Cells
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Cited by 3 •
2017
A modified density centrifugation gradient-based methodology was utilized to isolate epithelial cells from Rhipicephalus microplus gut tissue. Surface-bound proteins were biotinylated and purified through streptavidin magnetic beads for utilization in downstream applications.
09:58
Light-driven Enzymatic Decarboxylation
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Cited by 6 •
2016
We describe a protocol for the light-catalyzed generation of hydrogen peroxide — a cofactor for oxidative transformations.
FAQs
BirA uses ATP to activate biotin before transferring it to a particular lysine within an engineered acceptor peptide or protein domain. This defined acceptor site gives investigators greater control over where the affinity handle appears, which can help preserve the behavior or detectability of the labeled biomolecule during downstream analysis.
ATP supplies the activation step that enables BirA to react with biotin, while the acceptor lysine provides the covalent attachment site. These components connect the enzyme’s biochemical activity to the final labeling pattern. Changing the availability or placement of the acceptor sequence can therefore influence whether a target receives a useful, interpretable modification.
Targeted BirA labeling directs biotin to a specified lysine in an engineered acceptor peptide or domain. Other biotin ligases instead label molecules near the ligase inside living cells. The first strategy emphasizes defined modification of a chosen biomolecule, whereas proximity labeling records a local molecular environment for studying nearby components and relationships.
A common workflow places an engineered acceptor peptide or protein domain on the biomolecule of interest, supplies BirA and the conditions needed for ATP-dependent biotin activation, and then detects or isolates the modified material through streptavidin or avidin binding. The resulting handle can support protein purification, detection, or molecular tracking.
The strong interaction between biotin and streptavidin or avidin allows labeled molecules to be captured or detected after the enzymatic reaction. Depending on the study, this enables protein isolation, imaging, or tracking. These readouts can help reveal where a molecule is located, which components are associated with it, or how those relationships change.
Proximity-based labeling is useful when researchers want to examine molecules found near a biological target in living cells rather than modify only a predefined acceptor site. By labeling nearby molecules, the approach supports interaction mapping and analysis of local molecular composition. It can therefore contribute to studies of protein localization and dynamic molecular relationships.