样品实验方案
简要概述
准备并添加葡萄糖标准品和/或测试样品（50μL）
准备并添加葡萄糖分析工作溶液（50μL）
在37°C孵育10-30分钟
检测OD值=570nm左右的吸光度
溶液配制
1.储备溶液配制
除非另有说明，否则所有未使用的储备溶液应分成一次性等分试样，并在制备后储存在-20°C。避免反复冻融循环。
1.1Amplite 红色储备液（250X）：
将100μLDMSO（组分E）加入到Amplite Red底物（组分A）的小瓶中。应立即使用原液。任何剩余的溶液应等分并在-20℃重新冷冻。注意：避免反复冻融循环。注意：在硫醇如二硫苏糖醇（DTT）和2-巯基乙醇存在下，Amplite Red底物不稳定。反应中DTT或2-巯基乙醇的浓度应不高于10μM。 Amplite Red底物在高pH（> 8.5）下也不稳定。因此，反应应在pH 7-8下进行。建议使用提供的分析缓冲液（pH 7.4）。
1.2辣根过氧化物酶（HRP）储备液（10 U / mL）：
将1mL测定缓冲液（组分B）加入到辣根过氧化物酶（组分C）的小瓶中。注意：未使用的HRP溶液应分为单次使用的等分试样并储存在-20℃。
1.3葡萄糖氧化酶溶液（100 U / mL）：
将1mL测定缓冲液（组分B）加入葡萄糖氧化酶（组分D）的小瓶中。注意：未使用的葡萄糖氧化酶溶液应分为一次性等分试样并储存在-20℃。
1.4葡萄糖原液（800mM）：
将1mL测定缓冲液（组分B）加入葡萄糖小瓶（组分F）中。注意：未使用的葡萄糖溶液应分为单次使用的等分试样并储存在-20℃。
2.标准溶液配制
2.1葡萄糖标准溶液配制
通过将适量的800mM葡萄糖储备溶液稀释到测定缓冲液（组分B）中来制备葡萄糖标准品，以产生30μM的葡萄糖浓度。 然后在测定缓冲液（组分B）中进行1：3连续稀释，得到约10,3,1,0.3,0.1和0.03μM连续稀释的葡萄糖标准品。 包括非葡萄糖缓冲液对照作为空白对照。
3.工作溶液配制
表1.一个透明底96孔微孔板的分析工作溶液（2X）
| 组分 |
容积 |
| Amplite 红色储备液（250x） |
20ul |
| HRP储备液（10 U / mL） |
100ul |
| 葡萄糖氧化酶溶液（100 U / mL） |
100ul |
| 分析缓冲液 |
4.78ml |
| 总容积 |
5ml |
操作步骤
表2.固体黑色96孔微孔板中葡萄糖标准品和测试样品的布局。
GS =葡萄糖标准品（GS1-GS7）; BL =空白对照; TS =测试样品。
| BL |
BL |
TS |
TS |
| GS1 |
GS1 |
… |
… |
| GS2 |
GS2 |
… |
… |
| GS3 |
GS3 |
|
|
| GS4 |
GS4 |
|
|
| GS5 |
GS5 |
|
|
| GS6 |
GS6 |
|
|
| GS7 |
GS7 |
|
|
表3.每个孔的试剂组成
| 葡萄糖标准溶液 |
空白对照 |
测试样品 |
| 连续稀释：50μL |
测定缓冲液（化合物B）：50μL |
50ul |
注意：由于Amplite 红色底物（对非荧光产物）的过氧化，高浓度的葡萄糖（例如测试样品或标准品中的100μM）可能导致荧光信号减少。
葡萄糖测定
1.如表2和3中所述，将葡萄糖标准品和含葡萄糖的测试样品加入96孔黑色微孔板中。
2.将50μL葡萄糖测定工作溶液添加到葡萄糖标准品，空白对照和测试样品的每个孔中（表2），以使总葡萄糖测定体积为100μL/孔。 注意：对于384孔板，每孔加入25μL样品和25μL测定反应混合物。
3.将反应在37℃孵育10至30分钟，避光。
4.用荧光板读数器在Ex / Em = 530-570nm / 590-600nm（Ex / Em = 540 / 590nm）监测荧光强度。
数据分析
从空白标准孔获得的读数（RFU）用作阴性对照。 从其他标准的读数中减去该值，以获得基线校正值。 然后，绘制标准读数以获得标准曲线和方程。 该等式可用于计算葡萄糖样品。 点击使用在线线性回归计算器。
图1.使用Gemini酶标仪（Molecular Devices），在96孔黑色平板上用Amplite 荧光葡萄糖定量试剂盒测量葡萄糖剂量反应。
试剂应用文献
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Journal: Cellular and Molecular Life Sciences (2022): 1–17
Aspects {‘e}volutifs et environnementaux de la plasticit{‘e} ph{‘e}notypique chez deux Moronid{‘e}s, le bar Europ{‘e}en (Dicentrarchus labrax) et le bar ray{‘e} (Morone saxatilis)
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MicroRNAs regulate gene plasticity during cold shock in zebrafish larvae
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Journal: BMC genomics (2016): 922
Early nutritional programming in fish: tailoring the metabolic use of dietary carbohydrates
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Glucose metabolism and gene expression in juvenile zebrafish (Danio rerio) challenged with a high carbohydrate diet: effects of an acute glucose stimulus during late embryonic life
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Glucose overload in yolk has little effect on the long-term modulation of carbohydrate metabolic genes in zebrafish (Danio rerio)
Authors: Rocha, Filipa and Dias, Jorge and Engrola, Sofia and Gavaia, Paulo and Geurden, Inge and Dinis, Maria Teresa and Panserat, Stephane
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Inhibitors of fatty acid synthesis induce PPARα-regulated fatty acid β-oxidative genes: synergistic roles of L-FABP and glucose
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Journal: Journal of Biological Chemistry (2011): 44319–44325
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价格 2823
