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Related Experiment Video

Updated: Sep 9, 2025

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Published on: August 31, 2016

ALFA nanobody-guided endogenous labeling.

Zhe Wang^1,2, Fang Hu^1,3, Fudong Xue¹

¹Key Laboratory of Biomacromolecules, CAS Center for Excellence in Biomacromolecules, Institute of Biophysics, Chinese Academy of Sciences, Beijing, China.

Nature Chemical Biology

|August 29, 2025

Summary

This summary is machine-generated.

This study introduces ANGEL, a novel method using fluorescent nanobodies to detect successful ALFA tag knockins in cells. This technique enables efficient, high-throughput screening for genetic engineering applications.

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Last Updated: Sep 9, 2025

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Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

Published on: February 12, 2014

Area of Science:

Cell biology
Molecular biology
Biotechnology

Background:

Small peptide tags are useful for labeling endogenous proteins due to minimal interference.
Lack of inherent fluorescence in peptide tags complicates screening of modified cell lines.
High-throughput screening methods are needed for genetic engineering applications.

Purpose of the Study:

To develop a method for selectively illuminating cells with successful ALFA tag knockins.
To streamline high-throughput cell screening using fluorescence-activated cell sorting.
To enable versatile applications for endogenous protein labeling in native cellular environments.

Main Methods:

Development of antigen-stabilizing fluorescent protein-fused nanobodies (Nbs) targeting the ALFA peptide.
Engineering of ALFA nanobodies (NbALFA) for selective degradation in the absence of the ALFA peptide.
Utilizing fluorescence-activated cell sorting for high-throughput screening of knockin events.

Main Results:

Successful development of fluorescently labeled nanobodies (NbALFA) that are selectively degraded without the ALFA peptide.
Demonstrated a substantial increase in fluorescence intensity upon successful insertion of the ALFA peptide into the genome.
Established the ALFA Nb-guided endogenous labeling (ANGEL) technique for cellular applications.

Conclusions:

ANGEL provides an adaptable approach for selective cell illumination, facilitating efficient screening of genetic modifications.
The technique supports diverse applications including precise protein labeling, signal amplification, dynamic monitoring, and interactome analysis.
ANGEL enhances the utility of small peptide tags for endogenous protein studies within their native cellular context.