Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Quantitative Protein Determination
Protein Determination Using Trinitrobenzenesulfonic Acid in the Presence of Lipids

Reagents.

5% SDS solution (w/v) in Water.

2,4,6-Trinitrobenzenesulfonic acid (TNBS) (Eastman, white label) can be recrystallized from 1 M HCl. Prepare a 0.03 M solution in a tert-butanol — sec-butanol (9:1) mixture. The solution can be stored for 2 months at 0—2 °С.

Chloroform or diethyl ether.

Formic acid, 98%.

Procedure. A protein sample (0.04—0.5 µg) is treated with 20 µL of 5% SDS and dried under a stream of gas (N₂, СO₂, or air). Add 1 mL of 0.1 M potassium tetraborate and mix on a vortex mixer. Add 0.2 mL of the TNBS solution and mix again. Incubate for 30 min at 24—26 °С (10 min at 40°C) with occasional shaking (a vortex mixer can be used). Add 3 mL of chloroform or diethyl ether, mix immediately, and centrifuge for 1 min at 1000 g. Discard the organic phase containing Lipids and other impurities. Acidify the aqueous phase with 0.2 mL of HCl. Re-extract with 3 mL of chloroform or ether and discard the organic phase. Add 2.0 mL of 98% formic acid and heat at 100 °C for 20 min. After cooling the yellow-colored solution, measure the absorbance at 345 nm.

Note. Ovalbumin or BSA serves as standards. The determination error is ~10%. Other Methods for protein determination in the presence of lipids fail to yield reliable results. A similar method was developed for the determination of amines, Amino Acids, and Proteins in mixtures [257], as well as free amino groups in proteins [136]. The reaction of Amino Acids and Peptides with TNBS has been used for their detection in HPLC [381].