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。
Glu广泛存在于动物、植物、微生物和培养细胞中，不仅是组成蛋白质的20种氨基酸之一，而且通过转氨基作用参与多种氨基酸合成，是生物体内主要氨基来源之一。此外，Glu还是味精的主要有效成分，常用做食品添加剂以及香料生产。
测定原理：
利用专用提取液提取，然后用显色剂进行显色，显色后在570nm下进行测定。
注：本试剂盒仅限科研使用，不可用于临床诊断及其他用途。
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自备仪器和试剂
谷氨酸提取：
1. 细菌或培养细胞样品：先收集细菌或细胞到离心管内，离心后弃上清；按照细菌或细胞数量（10⁴个）：试剂一体积（ml）为500~1000：1的比例（建议1000万细菌或细胞加入2ml试剂一），超声波破碎细菌或细胞（冰浴，功率20％或200W，超声3s，间隔10s，重复30次）；8000g常温离心10min，取上清，置冰上待测。
2. 组织样品：按照组织质量（g）：试剂一体积(ml)为1：5~10的比例（建议称取约0.2g组织，加入2ml试剂一），进行冰浴匀浆。8000g常温离心10min，取上清，置冰上待测。
3. 血清（浆）或细胞培养液样品：按照血清（浆）或细胞培养液体积（ml）：试剂一体积(ml)为1：5~10的比例（建议取0.2ml血清（浆）或者细胞培养液加入2ml试剂一），进行冰浴匀浆。8000g常温离心10min，取上清，置冰上待测。
测定步骤：
1. 分光光度计预热30min以上，调节波长至570nm，蒸馏水调零。
2. 在有盖EP管中加入下列试剂：
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谷氨酸含量计算：
1. 标准条件下测定回归方程为y=0.0074x-0.5255；x为谷氨酸含量（μg/ml），y为吸光值。
2. 按照血清（浆）或者细胞培养液体积计算
谷氨酸含量(μg/ml)=[(ΔA+0.5255)÷0.0074×V1]÷(V3×V1÷V2）=1351×(ΔA+0.5255)
3. 按照蛋白浓度计算
谷氨酸含量(μg/mg prot)=[(ΔA+0.5255)÷0.0074×V1]÷(V1×Cpr)=135.1×(ΔA+0.5255)÷Cpr
4. 按照样本质量计算
谷氨酸含量(μg/g 鲜重)=[(ΔA+0.5255)÷0.0074×V1]÷(W×V1÷V2)=270.2×(ΔA+0.5255)÷W
5. 按照细菌或细胞密度计算
谷氨酸含量(μg/10⁴ cell)=[(ΔA+0.5255)÷0.0074×V1]÷(1000×V1÷V2)=0.27×(ΔA+0.5255)
V1：加入反应体系中样本体积，1ml；
V2：加入提取液体积，2ml；
V3：加入血清（浆）或细胞培养液体积，0.2ml；
Cpr：样本蛋白质浓度，mg/ml；
W：样本质量，g；
1000：细菌或细胞总数，1000万。
注意：
1. 该试剂盒仅适用于发酵液或组织中谷氨酸含量测定，检测下限为100μg/ml。
2. 标准曲线线性范围为：100μg/ml-600μg/ml。
3. ΔA线性范围为：0.01-2；若大于2则需要将上清液用试剂一稀释至适当倍数后测定，计算公式中乘以相应稀释倍数。
Glutamic acid (Glu) is widely distributed in animals, plants, microorganisms and cultured cells. As one of the 20 proteinogenic amino acids, it participates in the synthesis of various amino acids via transamination and serves as a major source of amino groups in organisms. In addition, glutamic acid is the principal active ingredient of monosodium glutamate and is commonly applied as a food additive and for spice production.
Assay Principle:
Samples are extracted with dedicated extraction solution followed by chromogenic reaction with color-developing reagent; absorbance is measured at 570 nm after color development.
Note: This kit is for research purpose only and shall not be used for clinical diagnosis or other applications.
Contents & Storage:
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Self-prepared Instruments and Reagents:
⁕Spectrophotometer (capable of absorbance measurement at 570 nm)
⁕Benchtop centrifuge, adjustable pipettes, 1 mL glass cuvettes, mortar
⁕Ice, distilled water
Glutamic Acid Extraction:
1. Bacteria or cultured cell samples: Collect bacteria or cells into centrifuge tubes and discard supernatant after centrifugation. Add Reagent Ⅰ at a volume ratio of 500–1000 : 1 (10⁴ cells : mL Reagent Ⅰ; recommended: add 2 mL Reagent Ⅰ per 10 million cells). Lyse cells by sonication on ice (power: 20% or 200 W, sonicate for 3 s, pause for 10 s, repeat 30 times). Centrifuge at 8000 g for 10 min at ambient temperature, collect supernatant and keep on ice pending assay.
2. Tissue samples: Homogenize tissue on ice with Reagent Ⅰ at a mass-volume ratio of 1 g tissue : 5–10 mL Reagent Ⅰ (recommended: weigh ~0.2 g tissue and add 2 mL Reagent Ⅰ). Centrifuge at 8000 g for 10 min at ambient temperature, collect supernatant and keep on ice pending assay.
3. Serum (plasma) or cell culture medium samples: Mix sample with Reagent Ⅰ on ice at a volume ratio of 1 : 5–10 (recommended: add 2 mL Reagent Ⅰ into 0.2 mL serum/plasma or cell culture medium). Centrifuge at 8000 g for 10 min at ambient temperature, collect supernatant and keep on ice pending assay.
Assay Procedures:
1. Preheat spectrophotometer for no less than 30 min, set wavelength to 570 nm and zero the instrument with distilled water.
2. Add listed reagents into capped EP tubes as follows:
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Calculation of Glutamic Acid Content:
1. The regression equation obtained under standard calibration: y=0.0074x-0.5255; Where: x= glutamic acid concentration (μg/ml); y= absorbance value.
2. Calculation based on serum (plasma) or cell culture medium volume
Glutamic acid (μg/ml)=[(ΔA+0.5255)÷0.0074×V1]÷(V3×V1÷V2）=1351×(ΔA+0.5255)
3. Calculation based on protein concentration
Glutamic acid (μg/mg prot)=[(ΔA+0.5255)÷0.0074×V1]÷(V1×Cpr)=135.1×(ΔA+0.5255)÷Cpr
4. Calculation based on fresh sample weight
Glutamic acid (μg/g fresh weight)=[(ΔA+0.5255)÷0.0074×V1]÷(W×V1÷V2)=270.2×(ΔA+0.5255)÷W
5. Calculation based on bacterial/cell density
Glutamic acid (μg/10⁴ cell)=[(ΔA+0.5255)÷0.0074×V1]÷(1000×V1÷V2)=0.27×(ΔA+0.5255)
V1: Sample volume added into reaction system, 1ml
V2: Volume of extraction solution (Reagent Ⅰ), 2ml
V3: Initial volume of serum/plasma or cell culture medium, 0.2ml
Cpr: Sample protein concentration, mg/ml
W: Fresh weight of sample, g
1000: Total number of bacteria/cells (ten million cells)
Notes:
1. This kit is only used for glutamic acid quantification in fermentation broth or tissue samples; the lower limit of detection is 100μg/ml。
2. Linear range of standard curve: 100μg/ml-600μg/ml。
3. Valid linear range of ΔA: 0.01-2; If measured ΔA>2, dilute the supernatant with Reagent 1 appropriately before testing, and multiply the final result by the corresponding dilution factor in calculation.
| 项目号 | 物理外观 | 组件 | 50T | 储存 |
| G1515930A | 液体 | 试剂一：液体 | 100 mL | 2-8℃. |
| G1515930B | 固体 | 试剂二：粉剂 | 1 EA | 2-8℃. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0636632 |
分析证书 | G1515930 |
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