Description
Biotin Ligase (also known as BirA, EC 6.3.4.15) can activate biotin to form biotinyl-5'-adenylate, and specifically transfer biotin to biotin receptor proteins (such as AviTag fusion proteins), thereby biotinylating the proteins.Component ListB1505408Component20µg2×20µgStorageB1505408ABiotin-Protein Ligase (1mg/ml)20µl2×20µl-80℃. Avoid freeze/thaw cycle.B1505408B10×Biotin Ligase Buffer A500µl1 mL-80℃. Avoid freeze/thaw cycle.B1505408C10×Biotin Ligase Buffer B500µl1 mL-80℃. Avoid freeze/thaw cycle.Activity and Purity The biological activity of Biotin-Protein Ligase is ≥ 7,500 Units/µg. Purity is > 95%, with no detectable protease activity.Storage Conditions1. Shipped on dry ice. After thawing, aliquot the product and avoid repeated freeze-thaw cycles.2. For long-term storage of Biotin-Protein Ligase, store at -80°C; for short-term use, store at -20°C; for frequent use, it can be stored at 4°C for a short period. When stored at 4°C, the enzyme can retain > 90% of its activity for three months.3. Store Ligase Buffer A and Ligase Buffer B at -20°C; repeated freeze-thaw cycles are acceptable.Example of Reaction SystemReactant ComponentVolume10×Biotin Ligase Buffer A2.5 µl10×Biotin Ligase Buffer B2.5 µlBiotin Ligase0.17 µlProtein (polypeptide) substrate1 - 5 µgdH₂Oup to 25 µl Total volume is 25 µl. Incubate at 25°C for 12 - 16 hours to fully biotinylate the substrate.Precautions1. The substrate of this product is AviTag fusion protein.2. If the substrate protein (polypeptide) needs to be stored at a lower temperature, the reaction temperature can be appropriately reduced and the enzymatic reaction time should be prolonged.3. Many common buffer components such as sodium chloride (>100 mM), glycerol (>5% W/V), and ammonium sulfate (>50 mM) can inhibit the activity of biotin ligase. When these reagents are necessary for the substrate protein (polypeptide), their concentrations should be reduced as much as possible.4. The substrate can contain Tris (pH 8.0), with an optimal concentration of 10 mM and no more than 50 mM.FAQsQ: How to prevent proteases from degrading the substrate?A: The biotin ligase of this product undergoes strict quality control and has no protease activity. If the substrate protein used for biotinylation is unpurified crude protein, it is recommended to add an appropriate amount of protease inhibitors to prevent degradation during the biotinylation process.Q: How to determine the optimal amount of this enzyme and reaction conditions for the reaction?A: Due to the great differences in the properties of different proteins and the certain uncertainty in biological enzyme reactions, the reaction conditions described in the manual are not completely applicable to all proteins. It is recommended that users conduct a pre-experiment: first take a small amount of protein, divide it into several equal amounts of substrates, add enzymes with different dilutions, corresponding amounts of Buffer A and B, react at 30°C for 1 h (or other time and temperature conditions), terminate the reaction with SDS-PAGE Loading Buffer, perform electrophoresis and transfer to an NC membrane for Western Blotting detection (after blocking with BSA or skim milk powder, directly incubate with commercially available HRP-Streptavidin, and develop color with DAB), compare the biotinylation situation, and select the optimal conditions. Since the binding force between Streptavidin and Biotin is extremely strong, only a small amount of biotinylated protein is needed for observation results when used for Western Blotting detection.Q: What should I do if the concentration of my substrate protein is quite low and it is not easy to concentrate during biotinylation?A: Add more enzyme and appropriately prolong the reaction time when conducting the enzymatic reaction with the same amount of substrate. However, there is a problem that cannot be ignored: the enzyme is a protein, and when the amount of enzyme increases, the amount of protein introduced into the system also increases. If subsequent experiments do not allow the presence of a large amount of heterologous proteins, then prolonging the reaction time will be the only choice.Q: Does detergent have any effect on the biotinylation reaction?A: Through experimental detection, 0.2% Tween 20 has no effect on the reaction. This product is only for experimental scientific research use. If any unit or individual uses this product for other special purposes specifically stipulated by the state such as clinical diagnosis and treatment, our company will not bear any responsibility