Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Quantitative Protein Determination
Protein Determination Using Sulfobromophthalein

Reagent. Mix 2.0 mL of 1 M HCl, 0.5 mL of 1 M citric acid, and 10 μL of sulfobromophthalein disodium salt solution (Baker Chemical) at a concentration of 50 mg/mL in Water.

2 M NaOH containing 1 mg/mL sodium deoxycholate (DOC).

Washing solution: a mixture of 1 M HCl + 1 M citric acid + 1 M NaOH (4:1:2).

Procedure. Place a protein sample (10 μL) in water or buffer into a 1.5 mL polypropylene centrifuge tube. Add an equal volume of 2 M NaOH containing DOC. Mix and leave for 1 h at room Temperature (if necessary for complete protein dissolution). Add 50 μL of the reagent, mix, and centrifuge (in an Eppendorf Model 3200 centrifuge at full speed, ∼12,000 g) for 1 min. Remove the unbound dye with a pipette. Add 0.5 mL of the washing solution. Centrifuge under the same conditions and discard the supernatant. Suspend the pellet in 0.5 mL of 0.1 M NaOH using a vortex mixer and measure the absorbance at 580 nm. A linear response is observed in the range of 0.5–10 μg of protein; increasing the dye concentration to 250 mg/mL maintains linearity up to 100 μg of protein.

Note. The method is not suitable in the presence of Triton X-100 (0.1%) and SDS (0.1%), as they interact with the dye. Reproducibility deteriorates when using a benchtop centrifuge operating at ∼2,000 g. Time intervals between steps are permissible. This method is recommended for protein determination in Tissues.