Practical Protein Chemistry - A. Darbre 1989

Analytical Methods
Determination of Protein Bound to a Solid Support
Matrix-Bound Protein

In Affinity Chromatography and solid-phase sequencing, it is often necessary to determine The amount of protein bound to an inert matrix such as agarose, dextrans, or Cellulose. In principle, this can be achieved by sample Hydrolysis followed by total nitrogen determination; however, the matrix may incorporate non-protein nitrogen, for instance, when BrCN activation is used or an N-containing spacer is introduced. Quantitative Amino acid analysis of the protein-matrix sample is frequently unsatisfactory due to high Background Interference from cleaved CARBOHYDRATES. Alternatively, one can perform a differential quantitative Analysis of the protein added to the matrix versus the uncoupled protein remaining after extensive washing. Nevertheless, Methods exist that enable rapid, direct quantification of immobilized protein, as described below (see also Section 12.3.3).

A modern Modification of the method [240, 398]. A known amount of Cu²⁺ is added to the carrier-bound protein. The amount of copper ions complexed with the protein is proportional to the quantity of immobilized protein and is determined by measuring the free copper ions via the reaction with diethyldithiocarbamate.

Reagents.

A. 0.167 M NaOH containing 3.4 g/L KNa tartrate tetrahydrate.

B. To 590 mL of Reagent A, add 10 mL of 0.2 M CuSO₄.

C. Sodium diethyldithiocarbamate (0.1 g) dissolved in 100 mL of a 0.025% crystalline BSA solution.

D. Suspension of AG1-X8 resin (Bio-Rad; 200–400 mesh, chloride form) in deionized Water (200 mg/mL).

Procedure. Wash the resin with the immobilized protein thoroughly to remove salts and free protein. Centrifuge a sample of the resin for 3 min at 300 g and measure the pellet volume. Add deionized water at a ratio ranging from 1:1 to 1:4 (depending on the amount of bound protein) to obtain a paste-like consistency. Take an aliquot of this paste (0.01–0.10 mL) and transfer it to a test tube. Dilute with distilled water to 1.5 mL. Add 1 mL of the reagent mixture (3–6 mL of Reagent B diluted to 10 mL with Reagent A). Mix well. After incubating for 15 min at room Temperature, add 5 mL of Reagent C. Mix and centrifuge at low speed. Measure the absorbance of the dark yellow supernatant at 446 or 486 nm against a water blank. Perform control measurements using untreated resin.

Calibration curve. To 0.5 mL of a series of solutions containing 50–800 µg of standard protein, add 1 mL of Reagent B. Mix, and after 15 min, add 1 mL of resin and 5 mL of Reagent C. Centrifuge and measure the absorbance as described above.