SpectBlue Protein Staining Reagent

1. Gel cleaning before staining

Place the SDS-PAGE gel in a clean container, add 300 mL~400 mL purified water, shake and wash for 5 minutes, repeat 3 times. If performing Native PAGE, rinse with purified water for 5 minutes.

Note: SDS inhibits the binding of the staining solution to the protein, so be sure to rinse the gel with sufficient purified water. If you use 200 mL purified water for cleaning, it is recommended to clean it for 10 minutes × 3 times. If the gel thickness is >1 mm or the concentration is >15%, it is recommended to clean it for 10 minutes × 3 times. If using large pieces of gel, please clean it with 5mL of purified water per square centimeter.

2. Staining

Shake SpectBlue staining solution evenly before use. Discard the purified water and add an appropriate volume of SpectBlue staining solution (appropriate to cover the gel). Place on a shaking table for staining for 15 to 30 minutes. After the staining is completed, discard the staining solution and add purified water to wash to remove the residual staining solution, and the results can be observed.

Note: If the protein content is low, the staining time can be appropriately extended. The actual staining time can be determined based on the degree of band visualization.

3. Decolorization

Add 200 mL of purified water for decolorization. The purified water can be replaced every 20 minutes for rinsing. Repeat 3 to 4 times to obtain a gel with extremely low background. Soaking the stained gel in purified water overnight can also achieve a good background removal effect and obtain clear protein bands.

Note: Microwave oven or water preheated to 50℃~60℃ can be used for decolorization to shorten the decolorization time. If large gels are used, please wash with 5 mL of purified water per square centimeter. Decolorization steps in microwave oven: add 100 mL purified water to each mini gel, after microwave for 30 seconds, gently shake it on a shaker for 5 minutes. This step can be repeated 2-3 times. Preheated water: add 100 mL of purified water preheated at 50℃ ~60℃ to a container with colloid and shake it gently for 5 minutes. This step can be repeated 2-3 times to completely clean the background.

• Cut the protein band to be analyzed with a tissue knife and destain it with 10%~30% ethanol or 20%~30% acetonitrile for 10 to 15 minutes.
• Wash with purified water and perform MS analysis.