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Cat. No.: BLKAPB-50 (for 50T)

Cat. No.: BLKAPB-250 (for 250T)

Cat. No.: BLKAPB-1250 (for 1250T)

Description

Biotin Labeling Kit for Avi-tag Protein with BirA is a kit that allows for quick and efficient biotin labeling of Avi-tag fusion proteins or polypeptides through the use of biotin ligase (BirA). Biotin-labeled proteins can be purified or detected using Streptavidin or Avidin-based methods. For example, biotin-labeled proteins can be purified using Streptavidin magnetic beads or detected using HRP-labeled Streptavidin.

The biotin ligase (Biotin-protein ligase, BirA) in this kit is derived from E. coli and is a recombinant protein expressed and purified using our proprietary protein technology platform. It is highly pure and active, with no additional tags, ensuring that it does not interfere with subsequent detection and purification of the biotin-labeled proteins. In the presence of ATP, BirA efficiently activates biotin (D-Biotin) to form biotinyl-5’-adenylate, and specifically attaches biotin to the lysine residues of the Avi-tag, thereby enabling quick and efficient biotin labeling of the target protein.

The Avi-tag is a short peptide tag consisting of 15 amino acids (GLNDIFEAQKIEWHE). In the presence of ATP and biotin, BirA attaches a biotin to the lysine residues of the Avi-tag, enabling biotinylation of the target protein. BirA's specific biotinylation of the Avi-tag offers several advantages. The Avi-tag is small and has minimal impact on the fusion protein, with biotinylation occurring specifically at lysine residues on the Avi-tag, resulting in high efficiency and repeatability. Biotinylation can be performed in vivo or in vitro, and biotinylated proteins exhibit high affinity for avidin or streptavidin, enabling applications such as immobilization, purification, and detection of the target protein. Compared to nonspecific labeling at random biotin sites in traditional biotinylation methods, BirA-catalyzed reactions are milder, simpler, have minimal impact on protein activity, and exhibit high enzyme efficiency and specificity.

The non-covalent interaction between biotin and avidin or streptavidin is known to be one of the strongest protein-ligand interactions. Avidin or streptavidin is a tetrameric protein capable of highly specific binding to four biotin molecules simultaneously, constituting a naturally occurring biomolecular interaction system. The biotin-avidin or biotin-streptavidin binding system, known as the biotin-avidin or biotin-streptavidin system, is a signal amplification labeling technology characterized by high affinity, sensitivity, specificity, and stability. It has been widely applied in various detection systems.

For in vitro biotinylation of Avi-tag proteins, based on a reaction system of 20μl or 50μl, with a protein (peptide) final concentration of 40μM, the small package of this product can be used for labeling 50 or 20 samples, the medium package for labeling 250 or 100 samples, and the large package for labeling 1250 or 500 samples.

Storage

Store at -20°C, valid for one year.

Notes

• It is recommended to aliquot Biotin Ligase Buffer B (10X) after the first thawing, as repeated freeze-thaw cycles may affect component activity.
• The amount of BirA, reaction temperature, and time can be adjusted according to different experiments, for example, BirA concentration can be adjusted appropriately between 0.2X and 2X.
• The protein (peptide) substrate of this product is a fusion protein or peptide with an Avi-tag.
• The final concentration of salts or reagents in the reaction solution has certain requirements, such as NaCl < 100mM, Glycerol < 5%, (NH4)2SO4 < 50mM, Tris < 50mM, otherwise it may inhibit the activity of biotin ligase.
• This product is for scientific research use by professionals only and is not intended for clinical diagnosis or treatment, food or drug use, or storage in residential settings.
• For your safety and health, please wear lab coats and disposable gloves during operation.