X-tal protocols

Protein assay: Bradford.

Accurate determination of the protein concentration is essential for most biochemical studies. Bradford protein assay is a rapid and sensitive method. Often interfering substances (detergents, buffers, etc) are essential in the protein sample. If the the concentration of the interfering substance is not known or the blank absorbance is to high, precipitation of the protein is a convenient method to remove interfering material.

Coomassie brilliant blue G-250 exists in three forms: cationic (470-nm red), neutral (650-nm green), and anionic (595-nm blue). The binding of the dye to the protein causes a shift in the absorption maximum of the dye from 470 to 595 nm, and it is the increase in absorption at 595 which is monitored.

Preparation of Bradford reagent:
This is a protocol used in specialized laboratories.
Follow the instruction written in the material safety data sheets.
Use at your own risk.
Dissolve 100 mg Coomassie Brilliant Blue G-250 in 50 ml 95% ethanol. Add 100 ml 85% (w/v) phosphoric acid and 50 ml water. Filter the solution through filter paper and store at -20°C.

Protein assay:
Pipet the reference protein solution (Bovine serum albumin), containing 1 to 25 µg protein, in a volume up to 900 µl, into a plastic micro cuvet.
Adjust the volume in the cuvet to 0.9 ml with appropriate buffer or water.
Add 0.2 ml of the bradford reagent to the cuvet, close the cuvet and mix.
Measure the absorbance at 595 nm after 2 min and before 1 hour, against a blank prepared from 0.9 ml of the appropriate buffer or water and 0.2 ml bradford reagent.
Plot the weight of the reference protein (1 to 25 µg bsa) against the corresponding absorbance resulting in a standard curve.
Determine the protein concentration of the unknown samples. [Bradford (1976)][Compton (1985)]

© 19-08-2018 Johan Zeelen