The detection of protein bands in the gel by Coomassie Blue Staining
depends on nonspecific binding of a dye, Coomassie Brilliant Blue
R, to proteins. The detection limit is 0.3-1 ug/protein band.
MATERIALS AND SOLUTIONS
Fixing Solution (100 ml)
50% Methanol ------------------------------- 50 ml of 100% Methanol
10% Acetic acid ----------------------------- 10 ml of Acetic
acid
Deionized, distilled H2O ---------------------- 40 ml
Use Fixing Solution only once.
Coomassie Blue Staining Solution (100 ml)
50% Methanol ------------------------------- 50 ml of 100% Methanol
0.005% Coomassie Brilliant Blue R ---------- 500 ul of 1% Coomassie Brilliant Blue R
10% Acetic acid ----------------------------- 10 ml of Acetic
acid
Deionized, distilled H2O --------------------- 40 ml
Dissolve the Coomavsie Brilliant Blue R in methanol before adding
acetic acid and water.
Use Coomassie Blue Staining Solution only once.
Destaining Solution (100 ml)
5% Methanol --------------------------------- 5 ml of 100% Methanol
7% Acetic acid ------------------------------- 7 ml of Acetic
acid
Deionized, distilled H2O ---------------------- 88 ml
PROCEDURES
1. Place the polyacrylamide gel(s) in a plastic box and fix the
gel with Fixing Solution for 5-10 min.
Agitate slowly on an orbital shaker.
2. Pour out Fixing Solution.
Stain the gel with Coomassic Blue Staining Solution for 1 hr.
Agitate slowly.
3. Pour out Coomassic Blue Staining Solution.
Rinse out with distilled water briefly.
4. Destain the gel with Destaining Solution for 1 hour to overnight.
5. Replace with new Destaining Solution occasionally. Agitate slowly.
6. Continue destaining until blue bands and a clear background
are obtained.
7. Store gels in 7% Acetic acid or distilled H2O.
Do not store gels in Fixing Solution. The protein bands will everaually
disappear.
8. To maintain a permanent gel record, the gel may be dried.
Place the gel between two cellophane membrane backing sheets that
are slightly larger than the gel.
Place this sandwich between
two sheets of Whatman 3MM filter paper and dry in a gel dryer
at 80oC for 60 min.
NOTES
Coomassie Brilliant Blue R binds nonspecifically to proteins.
Since the dye does not bind to the polyacrylamide gel, proteins
will be detected as blue bands surrounded by clear gel zones.