BirA酶工作液配制实例翻译

BirA酶工作液配制实例翻译
比拉酶促进生物素标记BSP位点的说明书实例，及计算方式，英文版翻译，有中文语法错误的轻参考原文。

Example calculation for determining the amount of BirA needed for your reaction:
For this example we will assume we are using a peptide substrate with AviTag™ of 15 kDa MW (note:the AviTag™ is 1.829 kDa MW).
1 nmol of a 15 kDa protein = 15µg; therefore 10 nmol = 150µg (2.5µg of BirA is recommended per 10nmol).
For a 1mg/mL solution of the substrate, 0.5mL would contain 500µg of that substrate.
- 500µg substrate/(150µg substrate/10 nmol) = 33.3 nmol of substrate to be biotinylated
- 33.3 nmol of substrate/(10 nmol of substrate/2.5µg BirA) = 8.325µg BirA
For best efficiency the substrate needs to be at 40µM concentration. In our example we have currently
0.5mL at 1mg/mL.
- 1mg/mL = 1000µg/mL = 66.6 nmol/mL = 66.6 µmol/L = 66.6µM.
We need to dilute the reaction solution to 40µM final.
- (66.6µM/40µM)(X/0.5mL); X = 0.83mL
So the final reaction mix would need to be brought up to 0.83mL with BiomixA (10X), BiomixB (10X),BirA and water (or your buffer of choice, see precautions below).
- 500µL of 66.6µM substrate + 83µL BiomixA + 83µL BiomixB + (164µL –XµL of BirA for 8.325µg*)water + XµL BirA = 0.83mL final reaction volume at 40 µM substrate concentration. (*BirA is offered at 1mg/ml or 3mg/mL concentrations).
用于确定反应所需的BirA量的示例计算：
对于此示例，我们将假设我们使用的肽基板与AviTag™分子量为15 kDa（注意：AviTag™™为1.829 kDa MW）。

15 kDa蛋白质的1 nmol = 15μg; 因此10 nmol = 150μg（推荐每10 nmol使用2.5μg的Bira酶）。

对于肽基板的1mg/mL溶液，0.5mL将包含该基板的500μg。

- 500μg基板/（150μg 基板/10 nmol）= 33.3 nmol 基板进行生物素化
500ug总量
15ug单量
×1nmol=33.3nmol
- 33.3 nmol 基板/（10 nmol 基板/2.5μg BirA）= 8.325μg BirA酶
33.3nmol总量
10nmol单量
×2.5ug=8.325ug BirA酶
为了获得最佳效率，底物需要浓度为40μM。

在我们的示例中，我们目前有0.5mL在1mg/mL.
- 1毫克/mL = 1000μg/mL = 66.6 nmol/mL = 66.6 μmol/L = 66.6μM。

我们需要稀释至40μM 浓度的反应解决方案。

-（66.6μM/40μ M）（X/0.5mL）;X = 0.83mL
66.6um 40um =X
0.5ml
；X = 0.83mL
因此，最终的反应组合将需要补充至0.83mL与生物混合A（10倍），生物混合B（10倍），BirA 和水（或您选择的缓冲区，请参阅下面的预防措施）。

- 500μL 66.6μM 底物+83μL BiomixA+83μL BiomixB+（164μL-BirA的XμL为8.325μg*）水
水+ XμL BirA= 0.83mL 最终反应体积，底物浓度为40 μM。

(*BirA的量基于提供的是1mg/ml还是3mg/ml得浓度）。

(*at 2.5µg BirA per 10 nmol AviTag’d substrate)
Note from the table above that it may be necessary to concentrate your substrate to meet the 40µMcriteria.
Buffer conditions that affect biotin ligase:
To ensure a rapid rate of biotinylation, it is recommended that the AviTag’d substrate be as close as possible to 40μM in the final reaction mix. The lower the substrate concentration in the reaction mix, the longer it will take for biotinylation to complete. For example, a substra te at 40μM may be completely biotinylated within 30-40 minutes, but at 4μM it will take more than 5 hours using the same amount of BirA enzyme. To complete the biotinylation reaction at 4μM in under an hour (i.e. 10 times faster), it is necessary to add 10 times more enzyme to the reaction mix. As it is, the amount of BirA enzyme added to the reaction mix may need to be varied to achieve biotinylation within an acceptable time-frame (see below).
影响生物素连接酶的缓冲液条件：
为确保快速生物素化，建议在最终反应混合物中，AviTag的底物尽可能接近40μM。

反应混合物中底物浓度越低，完成生物素化所需的时间就越长。

例如，40μM的底物可能在30-40分钟内完全生物素化，但在4μM时，使用相同量的BirA酶需要5个多小时。

为了在1小时内完成4μM的生物素化反应（即加快10倍），需要向反应混合物中添加10倍以上的酶。

实际上，可能需要改变添加到反应混合物中的BirA酶的量，以便在可接受的时间范围内实现生物素化（见下文）。

Typically, for every 10 nmol of substrate (at 40μM), we recommend 2.5μg of Bir A enzyme to complete the biotinylation in 30 - 40 minutes at 30°C or about 1 hour at room temperature. Reactions can be performed at 4°C but will require more time, overnight if it is convenient. This is acceptable because the ATP hydrolyzes more slowly at 4°C. Also, additional ATP in solution may be added to the reaction if there is a question of incomplete biotinylation due to ATP degradation. The other reactants are more than stabile enough to continue the biotinylation if this is the case.
通常，对于每10 nmol底物（40μM），我们建议使用2.5μg BirA酶在30°C下30-40分钟内或在室温下约1小时内完成生物素化。

反应可以在4°C下进行，但需要更多的时间，如果方便的话，需要过夜。

这是可以接受的，因为ATP 在4°C下水解更慢。

此外，如果由于ATP降解存在不完全生物素化的问题，溶液中的额外ATP可添加到反应中。

在这种情况下，其他反应物的稳定性足以继续生物素化。