Practical Protein Chemistry - A. Darbre 1989

Methods of solid-phase amino acid sequence analysis
Coupling methods
Monitoring coupling efficiency

12.4.5.1. Solubility test. Peptides are typically tested for solubility in the coupling buffer. The peptide is dissolved in the buffer, an aliquot containing 2 nmol of the sample is taken, and the sample is either dried in vacuo or applied directly to a TLC plate (Cellulose Cell 300, Macherey and Nagel). Chromatography is performed in the system: pyridine–butanol–acetic acid–Water (10:15:3:12, by volume). The plate is sprayed with a ninhydrin solution in acetone (3 g per 100 ml). Only a single spot should appear, with an intensity corresponding to 2 nmol of the peptide. If the sample volume exceeds 2.5 µl, salts should be removed by lyophilization. For macro-level structural analysis, to conserve material, this test should be performed using more sensitive Methods, such as HPLC or Amino acid analysis.

12.4.5,2. Monitoring the degree of coupling. Upon completion of the Condensation reaction, the supernatant is analyzed for residual (uncoupled) peptide. The sampled aliquot is hydrolyzed and subjected to amino acid analysis. If peptide is detected in the solution, the coupling is repeated under different conditions. If sufficient sample is available, it is desalted and reused in the condensation reaction.

Determination of the coupling yield. A portion of peptidyl-resin or Glass (10 mg) is washed with TFA (twice with 0.5 ml) to remove non-covalently bound peptide and dried in vacuo. The resin is mixed with 250 µl of a 12 M Hydrochloric acid–propionic acid (1:1) solution, placed in ampoules, evacuated, and incubated for 24–48 h at 110 °C [57] or for 2 h at 130 °C (in ampoules under nitrogen) [65]. The resin particles are then filtered off and discarded, the solution is evaporated, and the dry residue is dissolved in a citrate buffer for amino acid analysis (pH 2.2). Amino acid analysis is performed, and The amino acid yield is calculated. Hydrolysis of the peptidyl-polymer can also be carried out in a mixture of 12 M hydrochloric acid–acetic acid–phenol (2:1:1) under vacuum for 24 h at 110 °C [20].

Test runs on the Sequencer. The operating performance of the instrument and the purity of the Reagents and Solvents used can be verified using bradykinin or Insulin B-chain as reference standards (10–100 nmol of the sample is subjected to coupling) [78].