In which applications can biotinylated target proteins be utilized?

Biotin Labeling

What is Biotin?

Biotin is a small, soluble molecule produced by plants or prokaryotic organisms. It can form a non-covalent bond with avidin or streptavidin, exhibiting an exceptionally high binding affinity with a dissociation constant (Kd) of up to 10^-15 mol/L. The binding strength between biotin and avidin is one million times greater than that of antigen-antibody interactions.

Biotin consists of an imidazolidone ring and a thiophene ring. The imidazolidone ring is primarily responsible for binding to avidin, while the carboxyl group at the end of the thiophene ring side chain is used to conjugate with antibodies and other macromolecules.

Applications of the Biotin-Avidin System

Histochemistry

Immunofluorescence, immunoradiometric techniques, colloidal gold techniques

Molecular Biology，Gene expression regulation

Applications of Biotinylated Proteins: Examples

Antibody Affinity Maturation - ELISA

Immobilized Human SIRP-α Fc Chimera, Human (Cat. No. UA010005) at 1 μg/mL (100μL/well) can bind Biotinylated Human CD47 Fc Chimera (Cat. No. UA010007) with EC50 of 0.067 μg/ml.

Case Study: SIRP was immobilized, and biotinylated CD47 was serially diluted to assess the binding affinity between the two.

Case Study: In a study investigating the in vitro affinity maturation of human GM-CSF antibodies, the authors used a receptor inhibition ELISA to demonstrate the mutual exclusivity of GM-CSF binding to four antibodies and the GM-CSFR-alpha chain. The results showed that antibody binding to GM-CSF prevented the binding of GM-CSFR, with the parental MOR03929 exhibiting lower receptor inhibition (IC50: 50 pM).

CAR-T - FACS Detection

CD19 is one of the critical targets in CAR-T research. As a type I transmembrane protein of the immunoglobulin superfamily, CD19 is exclusively expressed in B cells and regulates B cell development through both B cell receptor-dependent and independent mechanisms.

Case Study: In a study monitoring the blood status of patients treated with approved CAR-T drugs, researchers used recombinant biotinylated Fc-tagged CD19 to label cells, followed by staining with phycoerythrin-labeled streptavidin. Classic T cell surface markers, including CD3, were then labeled with antibodies. After lysing red blood cells, flow cytometry analysis was performed.

Biopanning for Biopharmaceuticals

Case Study: In the study titled "Isolation and Characterization of Single-Domain Antibodies from Immune Phage Display Libraries," the authors aimed to isolate target-specific VHHs. During the biopanning process, streptavidin was pre-coated onto a 96-well plate, and biotinylated target proteins were bound to the streptavidin to immobilize the target. A phage library containing 10^12 phages was added to each well, and both unbound and bound phages were eluted. After multiple rounds of screening, suitable VHHs were obtained.

In addition to the above applications in ELISA, FACS, and Biopanning, biotinylated proteins are also widely used in SPR assays, BLI assays, purification, enrichment, and other experiments.

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Catalog number: UA010035

Purity: >95% by SDS-PAGE

Immobilized Human SIRPα Fc Chimera (Cat. No. UA010005) at 1 μg/mL (100 μL/well) can bind Biotinylated CD47 His Tag, Human with EC50 of 0.063 μg/ml.

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