适用仪器

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荧光酶标仪
激发：	490nm
发射：	525nm
cutoff：	515nm
推荐孔板：	黑色孔板

样品实验方案

简要概述

1. 准备BChE标准品，测试样品和染料工作溶液
2. 添加BChE标准品或测试样品（100 uL）
3. 添加BChE染料工作溶液（100 uL）
4. 在室温下孵育10-30分钟
5. 检测Ex / Em = 490/525nm的荧光强度

注意：开始实验前，在室温下解冻试剂盒所有成分。

 

溶液配制

储备溶液配制

1.Thiolite 绿色原液（200X）：将50 uL DMSO（组分E）加入小瓶Thiolite Green（组分A）中，制成200X Thiolite Green储备溶液。

2.丁硫代胆碱（BTC）储备溶液（100X）：将120 uL ddH2O加入BTC小瓶（组分C）中，制成100X BTC储备液。

3.丁胆碱酯酶（BChE）标准溶液（5 U / mL）：将50 uL含0.1％BSA的ddH₂O加入BChE标准品（组分D）小瓶中，制成5 U / mL BChE标准品溶液。

 

标准溶液配制

将20 uL的5U / mL BChE标准溶液添加到980 uL的测定缓冲液（组分B）中，以生成100mU / mL的BChE标准溶液（BS1），然后取100 mU / mL的BChE标准溶液（BS1），并在测定缓冲液（组分B）中进行1：3连续稀释，以得到系列换算的BChE标准溶液（BS2-BS7）。注意：减少的BChE标准溶液溶液，应在4小时内使用。

 

工作溶液配制

BChE染料工作溶液：将50 uL的200X Thiolite Green储备溶液和100 uL的100X BTC储备溶液添加到10 mL的测定缓冲液（组分B）中，使总体积为10.1 mL BChE染料工作溶液。 注意：避光。

 

实验步骤

表1.在透明的96微孔板中的BChE标准品和测试样品的布局。 BS = BChE标准品（BS1-BS7，100至0.13 mU / mL）; BL =空白对照； TS =测试样品

BL	BL	TS	TS
DS1	DS1	…	…
DS2	DS2	…	…
DS3	DS3
DS4	DS4
DS5	DS5
DS6	DS6
DS7	DS7

表2.  每个孔的试剂组成

Well	Volume	Reagent
DS1-DS7	100 uL	连续稀释 (100 to 0.13 mU/mL)
BL	100 uL	缓冲液 (Component B)
TS	100 uL	实验样品

1. 根据表1和表2提供的布局，准备BChE标准品（BS），空白对照（BL）和测试样品（TS）。对于384孔板，每孔使用25 uL的试剂代替100 uL。 注意：根据需要处理细胞或组织样品。

2. 将100 uL BChE染料工作溶液添加到BChE标准品，空白对照和测试样品的每个孔中，使总测定体积为200 uL /孔。 对于384孔板，将25 uL BChE工作溶液添加到每个孔中，总体积为50 uL /孔。

3. 在避光条件下，于室温下孵育反应10至30分钟。

4. 使用荧光酶标仪在Ex / Em = 490/525nm处检测荧光的增加。

 

图示

 

图1.使用Gemini荧光酶标仪在带有Amplite 荧光丁酰胆碱酯酶检测试剂盒的黑色96孔板上测定丁酰胆碱酯酶的剂量响应。 孵育10分钟可检测到低至0.1 mU / mL的丁酰胆碱酯酶。

 

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