Optogenetic Biotinylation
Optogenetic 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...
06:06
Optogenetic Functional MRI
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Cited by 15 •
2016
This protocol describes the steps and data analysis required to successfully perform optogenetic functional magnetic resonance imaging (ofMRI). ofMRI is a novel technique that combines high-field fMRI readout with optogenetic stimulation, allowing for cell type-specific mapping of functional neural circuits and their dynamics across the whole living brain.
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.
02:30
Optogenetic Control of Hippocampal Network Dynamics
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2025
This video demonstrates the method to use optogenetics to modulate hippocampal network dynamics by activating genetically modified light-sensitive interneurons in a mouse brain slice, enhancing theta oscillation synchronization and studying neural circuit behavior.
FAQs
Illumination activates the light-responsive domain within the genetically encoded fusion, switching on the attached biotin ligase. This control lets researchers determine when labeling begins, while the fusion’s selected cellular position determines where labeling occurs. The combination of light timing and molecular targeting supports measurements of protein neighborhoods as they change during cellular events.
ATP supplies the energy used by the biotin ligase to generate a reactive biotin intermediate. That intermediate then forms covalent attachments to nearby proteins, creating a stable biochemical label that can be recovered after illumination. Because the attachment is covalent, labeled proteins remain marked for subsequent enrichment and identification assays.
The method records proteins located near the selected molecule, organelle, or cellular region during the labeling period. Consequently, the resulting set describes a molecular neighborhood rather than an unrestricted cellular protein pool. This proximity-based information helps researchers examine local molecular interactions and compare how nearby proteins change under different cellular conditions.
A typical workflow begins by expressing the genetically encoded fusion that combines the light-responsive domain with the biotin ligase. Researchers then illuminate the selected cells or location to activate labeling. Afterward, biotinylated proteins are isolated with streptavidin and identified by mass spectrometry or analyzed with other assays, depending on the experimental goal.
Researchers can apply the method when protein organization must be examined at a defined place and time in living cells. Its light-dependent activation is useful for following molecular changes associated with signaling or trafficking, where neighborhoods may shift during a cellular process. The resulting labels provide spatially resolved information rather than only whole-cell measurements.
The approach can reveal which proteins occupy a selected molecular neighborhood during a defined illumination period. Streptavidin enrichment concentrates the labeled material, while mass spectrometry can identify its protein components; other assays can provide complementary analysis. These results support investigations of dynamic interactions, organelle-associated protein environments, signaling events, and trafficking-related changes.