氨基黑10B可用于检测印迹膜上的蛋白,转移的蛋白大于50ng/条带时,在淡蓝色的背景上可呈现深蓝色的条带,在测定内部序列时,对于蛋白质测序和蛋白质原位切割,氨基黑是最常用的染色剂。
氨基黑10B染色浸出液又称醋酸纤维薄膜电泳洗脱液,主要由碱、去离子水等组成,适用于氨基黑10B染色后的洗脱。可定量检测从醋酸纤维薄膜上洗脱出来的染料含量,与蛋白含量成正比关系。该试剂仅用于科研领域,不适用于临床诊断或其他用途。
自备材料
- 蒸馏水或去离子水、氨基黑10B染色液、氨基黑10B染色漂洗液
- 摇床
操作步骤(仅供参考)
1. 将印迹转移膜放在恰当容器中,蒸馏水水洗3次,每次5 min。
2. 浸入氨基黑10B染色液 (0.1%) 染色5 min或更长时间,染色过程中不时晃动染色皿。
3. 准备3个漂洗皿,加入氨基黑10B染色漂洗液,从染色液中取出薄膜条并尽量沥去染色液,按顺序投入漂洗液中反复漂洗,直至背景无色为止。
4. 将漂洗净的薄膜吸干,剪下将染色的蛋白区带放入相应的试管内,在白蛋白管内加入氨基黑10B染色浸出液4-6 mL,振摇数次,后置于37℃水箱中20 min,使染料充分浸出,用分光光度计测定各管在620 nm处的吸光度值。
注意事项
1. 本产品有一定腐蚀性,请小心操作。
2. 为了您的安全和健康,请穿实验服并戴一次性手套操作。
3. 试剂开封后请尽快使用,以防影响后续实验效果。
Amido Black 10B can be used to detect proteins on blotting membranes. When the transferred protein is greater than 50 ng per band, dark blue bands appear on a light blue background. Amido Black is the most commonly used stain for protein sequencing and in situ protein cleavage when determining internal sequences.
Amido Black 10B Staining Eluent, also known as Cellulose Acetate Membrane Electrophoresis Eluent, is primarily composed of alkali and deionized water. It is suitable for elution after Amido Black 10B staining. It can be used for the quantitative detection of dye eluted from cellulose acetate membranes, which is proportional to the protein content. This reagent is for research use only and is not intended for clinical diagnosis or other purposes.
Materials to Prepare
Distilled or deionized water
Amido Black 10B Staining Solution
Amido Black 10B Destaining Solution
Shaker
Procedure (For Reference Only)
Place the blotting transfer membrane in a suitable container. Wash with distilled water 3 times, 5 minutes each time.
Immerse the membrane in Amido Black 10B Staining Solution (0.1%) for 5 minutes or longer. Shake the staining dish occasionally during the staining process.
Prepare 3 destaining dishes containing Amido Black 10B Destaining Solution. Remove the membrane strip from the staining solution, drain off as much staining solution as possible, and successively immerse it in the destaining solutions. Rinse repeatedly until the background is colorless.
Blot the destained membrane dry. Cut out the stained protein bands and place them into corresponding test tubes. Add 4-6 mL of Amido Black 10B Staining Eluent to the tube containing the albumin band. Shake several times, then place in a 37°C water bath for 20 minutes to fully elute the dye. Measure the absorbance of each tube at 620 nm using a spectrophotometer.
Precautions
This product has certain corrosiveness. Please handle with care.
For your safety and health, please wear a lab coat and disposable gloves during operation.
Use the reagent as soon as possible after opening to prevent affecting subsequent experimental results.