Dissolve 100 mg of Coomassie G-250 in 50 ml of ethanol, mix well, add 800 ml distilled water and 100 ml of 85% phosphoric acid, make volume to 1lt with distilled water

https://en.wikipedia.org/wiki/Bradford_protein_assay

Hi Erel Hatan ,

As Antonina Kokisheva mentioned, first prepare the Bradford reagent. I usually let the 1 L preparation stir at least overnight and if possible over the weekend to make sure that everything has dissolved thoroughly.

After you have the reagent on hand, you will need to standardize your Bradford constant using varying concentrations of BSA from 0 - 10 ug. As you may already know, use the wavelength of 595 nm when using the spectrophotometer and measure the OD for the BSA samples. Using a linear regression equation, calculate the slope by graphing the OD values of the BSA. The calculated slope will be your Bradford constant, which you may use for downstream protein concentration calculations with the equation total mg/ml of protein = (absorbance at 595 nm/ (volume of sample in ul x Bradford constant)).

You may use this link if it helps you to calculate the unknown/ BSA standard: https://info.gbiosciences.com/blog/bid/164578/bradford-protein-assay-calculation-of-an-unknown-standard

Good luck and hope this helps!

Hello Erel Hatan

Coomassie Brilliant Blue G-250 binds stably to proteins in acidified solution. When the dye comes in contact with protein electron is donated to charged groups on the protein. This causes disruption of the protein structure resulting in the exposure of hydrophobic pockets to which the Coomassie Brilliant Blue G-250 binds with the help of its sulfonic acid groups binding to the positive amines. This stably bound Coomassie Brilliant Blue G-250 to the protein is the unprotonated blue form. So, upon binding to the protein there is a color change from red to blue.

Therefore, in addition to Coomassie Brilliant Blue G-250 you will need phosphoric acid and either methanol or ethanol which will provide the reactive functional group (OH).

So, you should dissolve 100 mg of Coomassie Brilliant Blue G-250 in 50 ml of 95% ethanol. Then add 100 ml of 85% (w/v) phosphoric acid. Dilute to 1 litre with deionized water.

Initially you will get a blue color just indicating dissolution of the free Coomassie Brilliant Blue G-250 dye. After adding phosphoric acid the color of the solution turns reddish-brown. This is Bradford reagent.

You can then follow the protocol for Bradford assay from the link below

https://www.ruf.rice.edu/~bioslabs/methods/protein/bradford.html

I hope this helps.

Best Wishes.