Hello.
I want to make a Bradford reagent but i have read that this reagent colud be red when it is cationic and blue when it is Anionic and green when it is neutral.
What is the effect of these colors on the results of protein concentration determination?
Is the color important?and what is the original color of the reagent after filtration?
Thanks for your suggestion.
The color of the Bradford reagent should be between red and brown and can be green in some cases which is not a big problem. However, it should not be blue without the addition of protein. It should only turn blue in presence of protein, which you can measure. For more details, read the original article attached.
The commercially prepared 1X Bradford reagent I use is brown, and turns blue in the presence of protein. I have also used a 5X commercial Bradford reagent, which was red, turned brown when diluted to 1X, then blue when added to protein.
We determine protein concentration by using Bio Rad Protein Assay where the reagent is red in colour.
It can also be prepared by the following step:
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 and is ready for use after filtering through Whatman paper.
when the reagent is prepared it is in acidic conditions, then the Coomassie G-250 present in the reagent is cationic, mainly doubly protonated, and is red, whereas in neutral conditions the dye is green, and the anionic form of the dye is blue. the dye is thus primarily protonated and red as in the reagent.
When the dye comes in contact with protein, the first electron is donated to charged groups on the protein. This disrupts the structure of the protein, resulting in exposure of hydrophobic pockets. The dye binds to these pockets, with the sulfonic acid groups binding to positive amines further there is attraction due to Van der Waals forces. The stably bound Coomassie G-250 is now blue and in the unprotonated form.
The change in colour from red to blue upon binding to the protein can now be measured spectroscopically, where the dye is as red in bradford reagent had an absorbance maximum of 470 nm which in the presence of protein now changed to the anionic blue form of the dye which shifts the Amax to 595 nm.
Since the amount of the blue anionic form is proportional to the amount of protein in the sample, the quantity of protein in a sample can now be measured directly by measuring the absorption at 595 nm. The neutral green form of Coomassie that has donated an electron but is not bound to protein does not interfere with measurement in the Bradford assay since it has an absorbance maximum of 650 nm.
All the best.
The Bradford reagent has Color is reddish brown with lambda max 470 nm. With proteins it develops blue color hence used for colorimetric estimation of proteins using Beer-Lambert law i.e. absorption is proportional to the concentration of the solution.Color is important because you can measure color intensity in a visible region and observe with naked eyes.
- Badges
- Science topic