High-Efficiency Avi-Tag Protein Biotinylation Kit

What is Biotin Labeling?

Biotin labeling is a widely used protein labeling technique that involves the conjugation of biotin molecules to specific proteins or substrates. This method leverages the high-affinity interaction between biotin and streptavidin (with a binding affinity of 10^-14 M to 10^-15 M) to achieve signal amplification and detection. Notably, this interaction remains stable under protein-denaturing conditions, such as in the presence of 1% SDS or 100 mM DTT.

Biotin labeling can be achieved through chemical or biological methods. Chemical biotinylation often lacks site specificity and may lead to protein inactivation. In contrast, enzymatic biotinylation, which offers high specificity and efficiency, is the preferred method.

BirA, a biotin ligase derived from Escherichia coli, specifically catalyzes the covalent attachment of biotin to the lysine residue within the Avi-tag (a 15-amino acid sequence: GLNDIFEAQKIEWHE). Kinetic studies have shown that the reaction rate using Avi-tag as a substrate is twice that of BirA's natural substrate, BCCP (biotin carboxyl carrier protein).

Figure 1: Schematic Diagram of Biotin Labeling Principle

Biotin labeling has been widely applied in various fields, including ELISA, SPR (surface plasmon resonance) molecular interaction detection, BLI (biolayer interferometry) molecular interaction detection, molecular biology, and histochemistry.

Steps for Biotin Labeling

The biotin labeling procedure is as follows:

1.Prepare the Reaction Mixture: Combine the Avi-tagged protein, buffers (UA070015), and BirA (UA070015) according to the following reaction system:

2.Incubation: Mix the components and incubate at 37°C. Aliquot 20 µL at 30, 60, and 90 minutes for analysis (additional time points may be taken if necessary).

3.Analysis: Use PullDown or other biotin quantification kits for analysis.

4.U-ACT PullDown Results:

10 nmol (40 µM) Avi-tagged protein substrate was labeled with 2 µg BirA at 37°C for 30 minutes. All sample lanes were loaded with 4 µg protein, respectively.

Streptavidin has four biotin-binding sites and may associate with 1 to 4 chains of the biotinylated protein. Therefore, the extent of protein biotinylation can be calculated based on the ratio of biotinylated protein to the original substrate protein.

Avi-Tagged Protein Biotinylation Kit

Efficient, Convenient, and High Purity

Product Code: UA070015

Product Name: Biotin Labeling for Avi-tag Protein

Specifications: 200 µg, 1 mg

BirA Enzyme Purity: >95% by SDS-PAGE & HPLC

Kit Components (200 µg Specification)