Exercise 4.3 - Coomasie Blue Staining of Protein Gels

LEVEL I

Materials

• Protein gel from Exercise 4.2
• 0.25% (w/v) Coomasie Brilliant Blue R 250 in methanol-water-glacial acetic acid (5-5-1), filtered immediately before use.
• 7% (v/v) acetic acid
• Commercial destaining unit (Optional)

Procedure

1. Place a gel prepared as in Exercise 4.2 in at least 10 volumes of Coomasie Blue staining solution for 2-4 hours. Rock gently to distribute the dye evenly over the gel.

2. At the conclusion of the staining, wash the gels with several changes of water.

3. Place the gels into a solution of 7% acetic acid for at least 1 hour.

4. If the background is still deeply stained at the end of the hour, move the gels to fresh 7% acetic acid as often as necessary.
   If a commercial destainer is available, this will decrease the time required for stain removal. Follow the manufacturer's directions for use of the destainer. 14

5. Place the gels into containers filled with 7% acetic acid as a final fixative.

6. Photograph the gels or analyze the gels spectrophotometrically.

Notes

Coomasie Brilliant Blue R 250 is the most commonly used staining procedure for the detection of proteins. It is the method of choice if SDS is used in the electrophoresis of proteins, and is sensitive for a range of 0.5 to 20 micrograms of protein. Within this range, it also follows the Beer-Lambert law and thus can be quantitative as well as qualitative. The major drawback is the length of time for the procedure and a requirement for destaining. Overstaining results in a significant retention of stain within the gel, and thus a high background stain, which might obliterate the bands. The length of time for staining must be carefully monitored, and can range from 20 minutes to several hours. If maximum sensitivity is desired, one should try 2 hours for a 5% gel and 4 hours for a 10% gel. Destaining must be monitored visually and adjusted accordingly.

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