BioReagent 级 ，适用于对基线干扰要求严格的色谱和分析工作流程。
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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。
天冬酰胺（Asn）是20种天然氨基酸之一，被所有生物用于蛋白质合成。在人类中是一种非必需氨基酸，因为它可以在体内由天冬氨酸和谷氨酰胺合成。
本试剂盒利用天冬酰胺酶使天冬酰胺分解成天冬氨酸和 NH₄⁺，接着在谷氨酸脱氢酶作用下使 NH₄⁺ 和 α-酮戊二酸反应，同时使 NADH 氧化，通过检测 NADH 在特异吸收波长 340 nm 处的下降量，进而计算出天冬酰胺（Asn）的含量。
组分与储存
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试剂处理注意事项
试剂一：
1.临用前 8000 g 4 ℃离心 2 min 使液体落入管底（可手动甩一甩）；
2.分别加 0.55 mL 蒸馏水溶解备用。用不完的试剂分装后 -20 ℃保存，禁止反复冻融，三天内用完。
试剂二：
1. 开盖前注意使粉体落入底部（可手动甩一甩）；
2. 分别加1.1 mL 蒸馏水溶解备用；
3. 保存周期与试剂盒有效期相同。
试剂三：
1. 开盖前注意使粉体落入底部（可手动甩一甩）；
2. 分别加1.1 mL 蒸馏水溶解备用；
3. 保存周期与试剂盒有效期相同。
试剂五：
1. 开盖前注意使粉体落入底部（可手动甩一甩）；
2. 加入1.1 mL 蒸馏水混匀备用；
3. 保存周期与试剂盒有效期相同。
标准品：
1. 标品浓度1 μmol/mL，此标准品不参与计算，仅用来验证试剂是否正常；
2. 保存周期与试剂盒有效期相同。
实验器材：
研钵（匀浆机）、冰盒（制冰机）、台式离心机、可调式移液枪、水浴锅（烘箱、培养箱、金属浴）、96孔板、离心管、酶标仪、蒸馏水（去离子水、超纯水均可）。
指标测定：
建议先选取 1-3 个差异大的样本（例如不同类型或分组）进行预实验，熟悉操作流程，根据预实验结果确定或调整样本浓度，以防造成样本或试剂不必要的浪费！
1、样本提取：
① 组织样本：0.1 g 组织样本（水分充足的样本建议取 0.5 g 左右），加 1 mL 的提取液研磨，粗提液全部转移到 EP 管中，12000 rpm，离心 10 min，上清液待测。
② 细菌/细胞样本：
先收集细菌或细胞到离心管内，离心后弃上清；取约 500 万细菌或细胞加入 1 mL 提取液，超声波破碎细菌或细胞（冰浴，功率 200 W，超声 3 s，间隔 10 s，重复 30 次）；12000 rpm 4 ℃离心 10 min，取上清，置冰上待测。
【注】：若增加样本量，可按照细菌/细胞数量（10⁴）：提取液（mL）为 500～1000：1 的比例进行提取。
③ 液体样品：澄清的液体样本直接检测，若浑浊则 12000 rpm，离心 10 min 取上清液待测。
2、检测步骤：(如用标准品来验证，请将下方样本替换为标准品进行操作)：
① 酶标仪预热 30 min 以上，调节波长至 340 nm。
② 所有试剂解冻至室温（25 ℃）。
③ 在 96 孔板中依次加入：
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【注】
1.若△A值在零附近，可增加样本取样质量 W 或增加样本加样体积 V₁（如增至 40 μL，则试剂四相应减少），则改变后的 W 和 V₁ 需代入计算公式重新计算。
2.若起始值 A₁ 太大如超过 2（如颜色较深的植物叶片，一般色素较高，则起始值相对会偏高），可以适当减少样本加样体积 V₁（如减至 10 μL，则试剂四相应增加），则改变后的 V₁ 需代入计算公式重新计算。
3.若△A的值大于 0.4，则需减少样本加样体积 V₁（如减至 10 μL，则试剂四相应增加），则改变后的 V₁ 需代入计算公式重新计算。
结果计算：
1、按照样本质量计算：
天冬酰胺（Asn）含量（μmol/g 鲜重）= [ΔA×V₂÷(ε×d)×10⁶] ÷ (W× V₁÷V) = 3.22×ΔA÷W
天冬酰胺（Asn）含量（μg/g 鲜重）= [ΔA×V₂÷(ε×d)×10⁶] ÷ (W×V₁÷V)×Mr = 425.4×ΔA÷W
2、按细胞数量计算：
天冬酰胺（Asn）含量（mmol/10⁴ cell）= [ΔA×V₂÷(ε×d)×10⁹] ÷ (500×V₁÷V) = 6.43×ΔA
天冬酰胺（Asn）含量（mg/10⁴ cell）= [ΔA×V₂÷(ε×d)×10⁹] ÷ (500×V₁÷V)×Mr = 849.5×ΔA
3、按照液体体积计算：
天冬酰胺（Asn）含量（μmol/mL）= [ΔA×V₂÷(ε×d)×10⁶] ÷V₁= 3.22×ΔA
天冬酰胺（Asn）含量（μg/mL）= [ΔA×V₂÷(ε×d)×10⁶] ÷V₁×Mr = 425.4×ΔA
4、按照蛋白浓度计算：
天冬酰胺（Asn）含量（μmol/mg prot）= [ΔA×V₂÷(ε×d)×10⁶] ÷ (Cpr×V₁÷V) = 3.22×ΔA÷Cpr
天冬酰胺（Asn）含量（μg/mg prot）= [ΔA×V₂÷(ε×d)×10⁶] ÷ (Cpr×V₁÷V)×Mr = 425.4×ΔA÷Cpr
V：加入提取液体积，1 mL
V₁：样本加样体积，0.02 mL
V₂：反应体系总体积，2×10⁻⁴ L
d：96 孔板光径，0.5 cm
ε：NADH 摩尔消光系数，6.22×10³ L/mol/cm
W：样本质量，g
500：细胞数量，万
Mr：天冬酰胺分子量，132.12
Cpr：蛋白浓度（mg/mL）
L-Asparagine (Asn) is one of the 20 naturally occurring amino acids and is used by all organisms for protein synthesis. In humans, it is a non-essential amino acid because it can be synthesized in the body from aspartic acid and glutamine.
This kit uses asparaginase to decompose asparagine into aspartic acid and NH₄⁺. Then, under the action of glutamate dehydrogenase, NH₄⁺ reacts with α-ketoglutarate, while NADH is oxidized. By measuring the decrease in NADH at the specific absorption wavelength of 340 nm, the content of L-asparagine (Asn) is calculated.
Contents & Storage
Note : Each component is individually packaged according to its storage temperature ( 2-80°C /-20 °C) upon shipment, with differentiated temperature‑controlled transportation to guarantee product performance. Orders containing materials requiring multiple temperature zones may be delivered in separate batches; please refer to the actual received goods. Upon receipt, store components separately at the temperatures specified in the component list. The shelf‑life is 3 months from the date of manufacture。 |
Reagent Handling Notes
Reagent I:
1.Before use, centrifuge at 8000 g at 4 ℃ for 2 min to bring the liquid to the bottom of the tube (you may also shake it down manually).
2.Add 0.55 mL distilled water to dissolve before use. Aliquot any unused reagent and store at -20 ℃. Do not freeze-thaw repeatedly. Use within three days.
Reagent II:
1.Before opening, make sure the powder falls to the bottom (you may shake it down manually).
2.Add 1.1 mL distilled water to dissolve before use.
3.The storage period is the same as the shelf life of the kit.
Reagent III:
1.Before opening, make sure the powder falls to the bottom (you may shake it down manually).
2.Add 1.1 mL distilled water to dissolve before use.
3.The storage period is the same as the shelf life of the kit.
Reagent V:
1.Before opening, make sure the powder falls to the bottom (you may shake it down manually).
2.Add 1.1 mL distilled water and mix well before use.
3.The storage period is the same as the shelf life of the kit.
Standard:
1.The standard concentration is 1 μmol/mL. This standard does not participate in the calculation and is only used to verify whether the reagents are working properly.
2.The storage period is the same as the shelf life of the kit.
Experimental Equipment
Mortar (homogenizer), ice box (ice maker), bench centrifuge, adjustable pipette, water bath (oven, incubator, metal bath), 96-well plate, centrifuge tubes, microplate reader, distilled water (deionized water or ultrapure water is also acceptable).
Assay Procedure(If verification is performed using a reference standard, replace the sample below with the reference standard for operation):
It is recommended to first select 1–3 samples with large differences (for example, different types or groups) for a preliminary experiment to become familiar with the procedure. Determine or adjust the sample concentration based on the preliminary experimental results to avoid unnecessary waste of samples or reagents.
1. Sample Extraction
① Tissue samples: Take 0.1 g tissue sample (for samples with high water content, about 0.5 g is recommended), add 1 mL extraction solution, and grind. Transfer the entire crude extract to an EP tube, centrifuge at 12000 rpm for 10 min, and use the supernatant for measurement.
② Bacterial/cell samples:
First collect the bacteria or cells into a centrifuge tube, centrifuge, and discard the supernatant. Take about 5 million bacteria or cells, add 1 mL extraction solution, and disrupt the bacteria or cells by ultrasonication (ice bath, power 200 W, ultrasonication 3 s, interval 10 s, repeat 30 times). Centrifuge at 12000 rpm at 4 ℃ for 10 min, collect the supernatant, and keep it on ice for measurement.
【Note】: If the sample amount is increased, extraction can be performed according to the ratio of bacterial/cell number (10⁴): extraction solution (mL) = 500–1000:1.
③ Liquid samples: Clarified liquid samples can be directly measured. If turbid, centrifuge at 12000 rpm for 10 min and use the supernatant for measurement.
2. Detection Procedure
① Preheat the microplate reader for more than 30 min and adjust the wavelength to 340 nm.
② Thaw all reagents to room temperature (25 ℃).
③ Add the following in sequence to a 96-well plate:
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【Note】
1.If the ΔA value is near zero, increase the sample weight W or increase the sample loading volume V₁ (for example, to 40 μL, then reduce Reagent IV accordingly). The changed W and V₁ must be substituted into the calculation formula for recalculation.
2.If the initial value A₁ is too large, such as exceeding 2 (for example, deeply colored plant leaves generally have relatively high pigment content, so the initial value may be relatively high), appropriately reduce the sample loading volume V₁ (for example, to 10 μL, then increase Reagent IV accordingly). The changed V₁ must be substituted into the calculation formula for recalculation.
3.If the ΔA value is greater than 0.4, reduce the sample loading volume V₁ (for example, to 10 μL, then increase Reagent IV accordingly). The changed V₁ must be substituted into the calculation formula for recalculation.
Result Calculation
1. Calculation Based on Sample Mass
L-Asparagine (Asn) content (μmol/g fresh weight) = [ΔA×V₂÷(ε×d)×10⁶] ÷ (W×V₁÷V) = 3.22×ΔA÷W
L-Asparagine (Asn) content (μg/g fresh weight) = [ΔA×V₂÷(ε×d)×10⁶] ÷ (W×V₁÷V)×Mr = 425.4×ΔA÷W
2. Calculation Based on Cell Number
L-Asparagine (Asn) content (mmol/10⁴ cell) = [ΔA×V₂÷(ε×d)×10⁹] ÷ (500×V₁÷V) = 6.43×ΔA
L-Asparagine (Asn) content (mg/10⁴ cell) = [ΔA×V₂÷(ε×d)×10⁹] ÷ (500×V₁÷V)×Mr = 849.5×ΔA
3. Calculation Based on Liquid Volume
L-Asparagine (Asn) content (μmol/mL) = [ΔA×V₂÷(ε×d)×10⁶] ÷V₁= 3.22×ΔA
L-Asparagine (Asn) content (μg/mL) = [ΔA×V₂÷(ε×d)×10⁶] ÷V₁×Mr = 425.4×ΔA
4. Calculation Based on Protein Concentration
L-Asparagine (Asn) content (μmol/mg prot) = [ΔA×V₂÷(ε×d)×10⁶] ÷ (Cpr×V₁÷V) = 3.22×ΔA÷Cpr
L-Asparagine (Asn) content (μg/mg prot) = [ΔA×V₂÷(ε×d)×10⁶] ÷ (Cpr×V₁÷V)×Mr = 425.4×ΔA÷Cpr
V: Volume of extraction solution added, 1 mL
V₁: Sample loading volume, 0.02 mL
V₂: Total reaction system volume, 2×10⁻⁴ L
d: Optical path length of 96-well plate, 0.5 cm
ε: Molar extinction coefficient of NADH, 6.22×10³ L/mol/cm
W: Sample mass, g
500: Cell number, ten thousand
Mr: Molecular weight of L-asparagine, 132.12
Cpr: Protein concentration (mg/mL)
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F1039746 |
分析证书 | A1521843 |
| ZJ26F1039745 |
分析证书 | A1521843 |
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