计算溶液所需的质量、体积或浓度。
Avi标签蛋白生物素标记试剂盒（BirA法）
有货
为什么选择此级别
EnzymoPure™,重组 级 提供 ≥95% 纯度，适用于对基线干扰要求严格的色谱和分析工作流程。
储存与运输
-80℃储存,避免反复冻融。特低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
生物素连接酶 (Biotin Ligase、BirA、EC 6.3.4.15)能活化生物素形成生物素酰 -5'-腺苷酸，将生物素特异地转移到生物素受体蛋白（如 AviTag 融合蛋白）上，使蛋白生物素化。
组分表
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活性及纯度
Biotin-Protein Ligase 生物活性≥7,500 Units/μg
纯度＞95% ，无可检测的蛋白酶活性。
储存条件
1. 干冰运送，解冻后分装，避免反复冻融；
2. Biotin-Protein Ligase 长期保存置于-80℃ , 短期内使用置于-20℃ , 频繁使用可短时置于4℃。在4℃条件下保存三个月可保持>90% 的酶活性；
3. Ligase Buffer A、B 储存于-20℃, 可反复冻融。
反应体系示例
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总体积 25µl，于 25℃孵育 12-16小时，使底物完全生物素化。
注意事项
1. 本产品的底物为 AviTag 融合蛋白。
2. 如底物蛋白（多肽）需保存在较低温度时，可适当降低反应温度并应延长酶促反应时间。
3. 氯化钠（>100 mM）、甘油（>5% W/V）、硫酸铵（>50 mM）等许多常见的缓冲液成分会抑制生物素连接酶的活性。当底物蛋白（多肽）确有必要使用这些试剂时，应尽量降低浓度。
4. 底物中可含有 Tris(pH 8.0) ， 最佳浓度为 10 mM，不超过 50 mM。
FAQs
Q：如何阻止蛋白酶降解底物？
A：本产品的生物素连接酶经严格质量控制，没有蛋白酶活性。如果用于生物素化的底物蛋白是未经纯化的粗提蛋白，建议加入适量的蛋白酶抑制剂， 以防止在生物素化的过程中发生降解。
Q：如何确定参与反应的最适本酶量及反应条件？
A： 由于不同蛋白性质差别很大、生物酶反应存在一定的不确定性，说明书中所述反应条件并不完全适用于所有蛋白。建议用户可以进行预实验：可先取少量蛋白，分成若干份相同量的底物，加入不同稀释度的酶，相应量的 Buffer A、B ，30℃ 1 h （或其它时间、温度条件）反应，以 SDS-PAGE Loading Buffer 终止反应后，电泳并转移至 NC 膜上进行 Western Blotting 检测（BSA 或脱脂奶粉封闭后直接以市售的 HRP-Streptavidin 孵育，DAB 显色），比较生物素化情况，选取最适条件。 由于 Streptavidin 与 Biotin 结合力极强，因而用于 Western Blotting 检测时，只需很少生物素化蛋白即可观察结果。
Q：如果我的底物蛋白浓度相当低且不容易浓缩，进行生物素化时该怎么办？
A:在进行相同底物量的酶促反应时加入更多的酶并适当延长反应时间。但有一个问题不容忽视，酶是蛋白质，在酶量增加的时候，引入体系中的蛋白量亦增加了，如果后续的实验不容许出现很大量杂蛋白，那么延长反应时间将是唯一的选择。
Q：去污剂对于生物素化反应有无影响？
A：经过实验检测，0.2%的 Tween 20 对反应没有任何影响。
该产品仅限于实验科学研究用，若有任何单位或个人将该产品用于临床诊断、治疗等其他国家专门规定的特殊用途，本公司概不承担任何责任。
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 List
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Activity and Purity
The biological activity of Biotin-Protein Ligase is ≥ 7,500 Units/μg.
Purity is > 95%, with no detectable protease activity.
Storage Conditions
1. 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 System
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Total volume is 25 μl. Incubate at 25°C for 12 - 16 hours to fully biotinylate the substrate.
Precautions
1. 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.
FAQs
Q: 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.
规格
别名
生物素链接酶 | 生物素蛋白标记试剂盒 | 生物素蛋白偶联试剂盒 | 生物素标记试剂盒 | 重组生物素连接酶
英文别名
Biotin Quick Labeling Kit with Biotin-LC-NHS | Biotin Protein Labeling Kit | Biotin Protein Conjugation Kit | Biotin Ligase | BirA | Biotin-Protein Ligase | BirA Enzyme
规格或纯度
EnzymoPure™, 重组, ≥95%, ≥7500 U/μg
英文名称
Biotin Labeling Kit for Avi-tag Protein with BirA
单位定义
底物蛋白（多肽）浓度为 40 µM 的条件下，于 25℃孵育 12-16小时 ，将 1 pmol 蛋白（多肽）生物素化所需的酶 量定义为一个活性单位。
纯度
≥95%
应用
Protein Labeling
储存与运输
储存条件
-80℃储存,避免反复冻融
运输条件
特低温运输
稳定性与储存
长期储存-80℃（1 年）；收货后建议分装，避免反复冻融。
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
溶液计算器
产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
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