Amido Black 10B (Naphthol Blue Black) is a highly toxic azo dye used for staining proteins on polyacrylamide gels and agarose gels.

Instructions
I. Preparation of reagents:
Configuration of mother solution and working solution: Amido Black 10B is usually provided in the form of solid dye. It needs to be dissolved in water or 50% methanol solution before the experiment. The commonly used concentration range is 0.1% to 0.5% (w/v), and the specific concentration can be adjusted according to the experimental requirements.
Note: When the dye is dissolved, a mixture of methanol and acetic acid (usually a 1:1 ratio) can also be used to improve the protein binding effect.
II. Protein staining on polyacrylamide gel:
Gel preparation: After completing the electrophoresis, remove the gel and rinse it briefly with distilled water to remove excess buffer.
1. Staining process: Immerse the gel in the prepared Amido Black solution. The staining time is usually 30 minutes to 1 hour, which can be shortened (15 minutes) according to the experimental needs.
2. After the dye binds to the protein, a dark blue or black complex will be produced.
3. Washing:
1) After staining, wash the gel thoroughly with a destaining solution (usually 7% acetic acid and 5% methanol, or 7% acetic acid alone) to remove unbound dye.
2) The washing time is usually 30 minutes to 1 hour, during which the destaining solution needs to be changed.
3) The protein bands should be clearly visible and contrast with the transparent gel background.
4 Visualization:
1) After washing, the staining results can be observed and recorded by conventional lighting or using a gel imaging system.
2) Using a scanner or gel documentation system, the staining intensity can be quantified at a wavelength of approximately 600–650 nm to analyze the staining intensity.
3. Protein staining on agarose gel:
The operation of agarose gel electrophoresis is the same as that of polyacrylamide gel, using the same staining and destaining methods.
4. Data analysis:
1. The concentration of the protein can be estimated by the staining intensity of the protein band. The size of a specific protein can be determined by comparison with a molecular weight standard.
2. Use a densitometer or imaging software to further quantify the intensity of the bands for comparative analysis between the experimental group and the control group.
Notes:
1. Toxicity: Amido Black 10B is a toxic dye. Appropriate safety measures should be taken during operation, such as wearing gloves, lab coats, and goggles, and ensuring that the operation is carried out in a well-ventilated environment.
2. Proper disposal: Properly dispose of Amido Black solution and gel according to laboratory safety procedures.
3. Staining time: Staining time may vary depending on the content and size of the protein. Care should be taken to control the staining time and avoid overstaining.
4. Background staining: If destaining is not thorough, background staining may occur, affecting the visibility of the bands. Be sure to destain thoroughly.