ProtoBlue Safe Protocol

Prepare Working Solution
1. Gently invert the bottle several times to resuspend colloidal dye particles that settle out on standing.
2. Add 1 part ethanol to 9 parts staining solution while stirring. (Standard denatured ethanol is fine). A 20ml to 50ml volume of working solution is typically prepared, depending on the shape and size of the staining container. To preserve solution, we recommend a plastic staining container just big enough to hold the gel.

Procedures for Gel Staining
1. Prepare ProtoBlue Safe Working Solution (above).
2. Wash the gel 3 times with 5 minutes each with deionized water on an orbital shaker. Decant wash solution.
3. After the last wash, add enough ProtoBlue Safe Working Solution to completely cover the gel.
4. Bands containing more than 1μg of protein will be detected within 15 minutes. For full sensitivity incubate the gel in the stain for at least 4-5 hours. Longer incubations in the stain will not adversely affect the gel or the staining sensitivity.
5. Remove the stain and wash the gel in deionized water. Incubating the gel in water increases the sensitivity of detection by reducing the background to crystal clear. The gel is stable in water for up to a week without loss of sensitivity. There is no need to store the gel in a salt solution.

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