Practical Protein Chemistry - A. Darbre 1989

Methods for Solid-Phase Amino Acid Sequence Analysis
Materials
Reagent and Buffer Solutions for the Sequenator

Reagents and Solvents purified as described above should be used.

Buffer No. 1 for the Condensation reaction: pyridine — N-methylmorpholine — trifluoroacetic acid. Dissolve 28 mL of N-methylmorpholine in 190 mL of Water, add 3.75 mL of TFA, and bring the volume to 200 mL with water: the pH of the resulting solution is 8.1. Add pyridine to this buffer at a ratio of (2:3), after which the pH of the final solution is 7.9. Use freshly prepared buffer and store under nitrogen.

Buffer No. 2 for the condensation reaction (anhydrous): DMF — triethylamine. Add 2.5 mL of triethylamine to 97.5 mL of DMF.

Buffer No. 3 for the condensation reaction (for macroscale analysis using DABITC — FITC): pyridine — N-ethylmorpholine — trifluoroacetic acid — DMF. Dissolve 4.5 mL of N-ethylmorpholine in 22.5 mL of water under nitrogen. Add a few drops of TFA, then water to a volume of 30 mL (the pH of the resulting solution is 9.1). To 10 mL of this buffer, add 20 mL of DMF and 20 mL of pyridine, and degas. Store under nitrogen.

Solution of methyl isothiocyanate (MITC) in acetonitrile. Dissolve 1 g of MITC in 1 mL of acetonitrile.

Phenyl isothiocyanate (FITC) solution: 10% solution of FITC in acetonitrile. Add 5 mL of FITC to 50 mL of acetonitrile under nitrogen.

FITC solution No. 2 (for Microscale analysis with DABITC — FITC): 5% FITC in DMF. Add 1 mL of FITC to 19 mL of DMF under nitrogen.

DABITC solution (for microscale analysis): 0.5% DABITC in DMF. Add 100 mg of DABITC to 20 mL of DMF under nitrogen. Prepare a fresh solution every 24 h.

Solvent for macroscale analysis: 0.2% triethylamine in methanol. Add 1 mL of triethylamine to 500 mL of methanol under nitrogen.