I am currently conducting a protein assay with the Bio-Rad Protein Assay Dye Reagent Concentrate, it's active ingredient is Coomassie Brilliant blue G-250. When distilled water is added to the concentrated dye according to the ratio given on the Bio-Rad manual (1 part of concentrated dye 4 parts of dH2O), the colour changed from its darkish-red to a lighter brown colour. Then, to test how the water affects the dye, I continued adding more distilled water. As more water is added, the colour changed from light brown to green, then from green to aqua, then aqua to blue. When the colour stopped changing, no more water is added. The whole volume of the water-dye solution is now around 50mL, and it appears to be light blue. Before & during & after adding distilled water, the water-dye solution has remained at a pH of 1.5 (due to the presence of phosphoric acid in the dye). Is this colour change normal when water is added? This may or may not be related to the of the expiration by 1.5 years from its 1 year shelf life. Thanks.
Hello Ryan,
If you want to know whether your batch of Bio-Rad protein assay reagent is still good, just try to perform a standard curve with BSA according to the instructions of the manufacturer. It it works, don't worry about colour changes upon diluting with increasing volumes of water (although you may find it lots of fun, of course)
If you are going to have fun, go all the way.
Article The Coomassie chronicles: Past, present and future perspecti...
The effect of slight changes of pH on the color of Coomassie Blue G-250 is described in this Wikipedia article.
https://en.wikipedia.org/wiki/Coomassie_Brilliant_Blue
Since diluting the dye solution with water would be expected to raise the pH slightly, that is probably why the color changes.
For a compatibility table, see the last page of this document:
https://www.thermofisher.com/document-connect/document-connect.html?url=https%3A%2F%2Fassets.thermofisher.com%2FTFS-Assets%2FLSG%2Fmanuals%2FMAN0011203_CoomassiePlus_Bradford_Asy_UG.pdf&title=VXNlciBHdWlkZTogIENvb21hc3NpZSBQbHVzIChCcmFkZm9yZCkgQXNzYXkgS2l0
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