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。
3,3',5,5'-四甲基联苯胺 (TMB)是非常优越的酶免试验显色剂，能溶解于多种有机溶剂和双蒸水中，为稳定的无色溶液，与适量过氧化脲或双氧水与缓冲液混匀后与过氧化物酶作用产生清晰的蓝色产物，极易观察。由于其灵敏度高，配制后的工作液稳定，加上无致癌特性，被广泛用于酶联免疫 (ELISA)试验和尿试纸的检测反应等，TMB主要应用于酶联免疫吸附实验 (ELISA)，经典的方法是采用A、B液分开配制，使用前再混合的方法，并且长期存放稳定，本试剂一般情况下为无色，有时略显浅蓝色或浅黄色。
TMB底物显色试剂盒 (ELISA, HRP显色)主要由TMB显色液、TMB Buffer、TMB氧化剂组成，其特点是：
1、灵敏度高；2、稳定性好，显色终止后读数稳定；3、背景低：底物溶液在450nm检测时OD值一般小于0.05。该试剂仅用于科研领域，不宜用于临床诊断或其他用途。
自备材料：
1、酶标仪、酶标板或96孔板、恒温箱 (备选)
2、(备选)终止液：盐酸或硫酸等
操作步骤 (仅供参考)：
1、用适当的干净容器 (用纯净水反复冲洗)，取试剂 (A)、(B)、(C)，按A: B: C= 5000: 5000: 4的比例混合，即为TMB显色工作液，待达到室温后即可使用。
2、加液：加完HRP结合物并温育一定时间，洗板3-5次，每孔加TMB显色工作液100 μl，在室温(15-25℃)或37℃下温育10-30分钟。
3、(选做)终止：加等体积的1M盐酸或硫酸溶液终止反应，孔中反应液由蓝色变为黄色。
4、读数：30 min内在450 nm处读数。
注意事项：
1、本试剂盒给予的TMB氧化剂多于实际使用量，请按比例使用。
2、TMB氧化剂易挥发，请注意密闭保存，以免效率下降，一旦开封请尽快使用。
3、TMB氧化剂有腐蚀性，请勿直接接触于人皮肤、毛发等。
4、如果出现高的反应背景或沉淀，表明TMB底物反应过于强烈。
5、终止反应不是必须步骤，如不加终止液应显色完成后立即检测。
6、一般市售的浓盐酸为11.6M，浓硫酸为18.3M，但应注意其挥发性。
7、为了避免产生沉淀，可在终止后马上读数，或者进一步稀释一抗和/或HRP结合物，再次进行试验。
8、为了您的安全和健康，请穿实验服并戴一次性手套操作。
9、试剂开封后请尽快使用，以防影响后续实验效果。
3,3',5,5'-Tetramethylbenzidine (TMB) is an excellent chromogenic reagent for enzyme immunoassays. It is soluble in various organic solvents and double-distilled water, presenting as a stable colorless solution. When mixed with an appropriate amount of urea peroxide or hydrogen peroxide and buffer solution, it reacts with peroxidase to produce a distinct blue product that is extremely easy to observe. Due to its high sensitivity, stable working solution after preparation, and non-carcinogenic properties, TMB is widely used in enzyme-linked immunosorbent assay (ELISA) and urine test strip detection reactions, etc. TMB is mainly applied in ELISA; the classic method involves separate preparation of Solution A and Solution B, which are mixed before use and remain stable for long-term storage. This reagent is generally colorless, and sometimes slightly light blue or pale yellow.
TMB Substrate Chromogenic Kit (for ELISA, HRP Chromogenesis) is mainly composed of TMB Chromogenic Solution, TMB Buffer, and TMB Oxidant, with the following characteristics: 1. High sensitivity; 2. Good stability with stable reading after chromogenic termination; 3. Low background: the OD value of the substrate solution detected at 450 nm is generally less than 0.05. This reagent is for research use only (RUO) and not intended for clinical diagnosis or other applications.
Materials to Be Prepared by the User
1. Microplate reader, microtiter plate or 96-well plate, incubator (optional)
2. Stop solution (optional): Hydrochloric acid, sulfuric acid, etc.
Operating Procedures (For Reference Only)
1. Take Reagent A, B and C in a suitable clean container (rinsed repeatedly with pure water), and mix them at a volume ratio of A: B: C = 5000: 5000: 4 to prepare the TMB chromogenic working solution, which can be used after reaching room temperature.
2. Solution addition: After adding HRP conjugate and incubating for a certain period, wash the plate 3-5 times, add 100 μl of TMB chromogenic working solution to each well, and incubate at room temperature (15-25℃) or 37℃ for 10-30 minutes.
3. Termination (optional): Add an equal volume of 1 M hydrochloric acid or sulfuric acid solution to terminate the reaction; the reaction solution in the wells will change from blue to yellow.
4. Reading: Measure the absorbance at 450 nm within 30 minutes.
Precautions
1. The amount of TMB Oxidant provided in this kit exceeds the actual usage; please use it in strict accordance with the specified ratio.
2. TMB Oxidant is volatile; please store it in a sealed container to avoid activity loss, and use it as soon as possible after opening.
3. TMB Oxidant is corrosive; do not allow direct contact with human skin, hair, etc.
4. High reaction background or precipitation indicates an excessively intense TMB substrate reaction.
5. Reaction termination is not a mandatory step; if no stop solution is added, perform the detection immediately after the completion of chromogenesis.
6. Commercially available concentrated hydrochloric acid is generally 11.6 M and concentrated sulfuric acid is 18.3 M; attention should be paid to their volatility.
7. To avoid precipitation, perform the reading immediately after termination, or further dilute the primary antibody and/or HRP conjugate and repeat the experiment.
8. For your safety and health, wear a lab coat and disposable gloves during operation.
9. Use the reagents as soon as possible after opening to prevent adverse effects on subsequent experimental results.
| T1509497 | Component | 2×50 mL | 2×100 mL | Storage |
| T1509497A | TMB显色液 | 50 mL | 100 mL | 2-8℃. Store in the dark. |
| T1509497B | TMB Buffer | 50 mL | 100 mL | 2-8℃. |
| T1509497C | TMB氧化剂 | 100 μl | 2×100 μl | 2-8℃. Store in the dark. |
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| 批号(Lot Number) | 证书类型 | 货号 |
|---|---|---|
 ZJ26F0838120 |
分析证书 | T1509497 |
| ZJ26F0434177 |
分析证书 | T1509497 |
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