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.

The above information is based on published literature. Experimental procedures should be appropriately modified to meet specific research demands.

DMSO: 30.83 mg/mL (50.01 mM), Sonication is recommended.