I am trying to prepare Bradford reagent in lab. When I'm adding 95% ethanol to commisse blue G250 dye to dissolve it, the solution is turning blue. Does it indicate to any problem in Bradford reagent preparation. Please suggest any solution!
This is the recipe for Bradford dye:
Dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol. Add 100 ml of 85% (w/v) phosphoric acid. Once the dye has completely dissolved, dilute to 1 litre with deionised water.
Once the ethanol solution is mixed with phosphoric acid and diluted with water, it should be brownish.
Follow the correct preparation method as suggested by dr. adam. Also, check this out:
https://zageno.com/l/ts-bradford-assay
Samples are dark blue: High alkaline concentrations: This raises the pH above the Bradford reagent’s limit. Dilute or dialyze your sample
Dear Dipanshu, thank you for asking this interesting technical question. I think an initial blue color just indicates dissolution of the free Coomassie Brilliant Blue G-250 dye. After addition of phosphoric acid the color of the reagent should be red-brown. The blue color comes back in the presence of protein. For more information please have a look at the original reference published by M. M. Bradford in 1976 which is freely available as public full text:
A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding
(see attached pdf file)
Good luck with your work and best wishes, Frank Edelmann
Frank T. Edelmann - I do not know these things, but I have read your answer, because your answers are written well, and it seems that they are useful at the level of contents.
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