Exercise 4.3 - Coomasie Blue Staining of Protein Gels
- 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)
- 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.
- At the conclusion of the staining, wash the gels with
several changes of water.
- Place the gels into a solution of 7% acetic acid for at
least 1 hour.
- If the background is still deeply stained at the end of
the hour, move the gels to fresh 7% acetic acid as often as
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.
- Place the gels into containers filled with 7% acetic
acid as a final fixative.
- Photograph the gels or analyze the gels
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
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Cell Biology Laboratory Manual
Dr. William H. Heidcamp, Biology Department, Gustavus Adolphus College,
St. Peter, MN 56082 -- firstname.lastname@example.org