I prepared Bradford reagent based on the following, but at the end it was green instead of brown. What could be the reason?
"First, dissolve 100mg of Coomassie Brilliant Blue G- 250 in 50ml of ethanol or methanol and then add 100ml 85% (w/v) phosphoric acid and once the dye has been completely dissolved, dilute it to 1 litre with deionised water."
Color depends upon nature of the solution , i.e. acidic , Neutral & anionic Condition.
1. Dissolve 50mg of Coomassie Blue G250 in 50ml of methanol.
2. Add 100ml of 85% H3PO4 to the solution from step 1.
3. Add the solution from step 2 into 500ml of H2O and mix.
4. Filter to remove and precipitates.
5. Add an additional 350ml of H2O.
6. Store at 4oC.
Under acidic conditions, Coomassie G-250 is cationic, mainly doubly protonated, and is red, whereas in neutral conditions the dye is green, and the anionic form is blue.
You must check the pH of the reagent because the color depends on the pH. If you don't have neutral, cationic, or anionic colors, you probably have a pH corresponding to a transition zone. Even oxidative reactions in reagents cause color changes.
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