Overview:
- Coomassie Brilliant Blue is a protein stain, which is frequently used to visualize protein on acrylamide gels.
- Chemically it is a disulfonated triphenylmethane compound.
- Coomassie Brilliant Blue is available in two forms, R-250 (red-tinted) and G-250 (green-tinted). G-250, also called colloidal coomassie dye, has additional two methyl groups.
- Both types of Coomassie Brilliant Blue dye reversible stains proteins, therefore, are very useful in protein chemistry. In acidic conditions, the dye binds to proteins primarily through basic amino acids (primarily arginine, lysine, and histidine), leading to shifting from reddish-brown to bright blue (absorption maximum – 595 nm).
- They are highly sensitive and can detect as little as 0.5 µg protein / cm2 of gel.
- R250 form is insoluble in water, but can be easily dissolved in methanol or ethanol, whereas, G250 form is soluble in water, methanol, and ethanol.
REQUIREMENTS
Reagents and solutions
Coomassie Brilliant Blue R-250
Methanol
Glacial acetic acid
Double distilled water / Deionized water
Equipment and disposables:
Conical flask / Beaker
Measuring cylinder
Weighing balance
Fume hood
Composition
0.3 % Coomassie Brilliant Blue R-250 (by weight)
45 % Methanol (by volume)
10 % Glacial acetic acid (by volume)
Objective
Preparation of Coomassie Brilliant Blue R-250 Solution for Detection of Protein on Acrylamide Gel
PROCEDURE
Step 1: Prepare a 100 ml solution containing 45 % Methanol and 10 % Glacial acetic acid in water.
To prepare this, take 45 ml water in a measuring cylinder and add 45 ml methanol and 10 ml glacial acetic acid. Mix.
Step 2: Weigh out 0.3 g of Coomassie Brilliant Blue R-250 in a conical flask (capacity 250 ml). Add the Methanol/Glacial acetic acid/water solution. Mix by swirling the conical flask.
Storage:
Store the solution at room temperature in a tightly sealed bottle.
