Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Determination of protein attached to a solid support
Determination of amino groups on hydrophilic matrices

This modified version [4] of the method [257] is designed for hydrophilic matrices, as the reaction proceeds exclusively in an aqueous medium.

A specific volume of gel containing no more than 4–5 µmol of amino groups is diluted with Water to 9 ml of 0.1 M K2B4O7, and 1 ml of 0.01 M trinitrobenzenesulfonic acid is added. Simultaneously, a reference solution is prepared using the same volumes of Reagents but without the gel. The gel is allowed to settle or is centrifuged. 1 ml of the supernatant is diluted with 5 ml of 0.1 M K2B4O7, and 0.5 ml of 0.03 M Glycine is added. In the blank test, 0.5 ml of water is added instead of the glycine solution. After standing for 25 min at 25 °C, 10 ml of cold methanol is added, and the absorbance is measured at 340 nm.

The concentration of amino groups on the gel is determined from the difference in absorbance between the test sample and the control solution (ε = 12400). The determination error is <10%.



Last update: 06/08/2026

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