Practical Protein Chemistry - A. Darbre 1989

Traditional strategy for determining protein structure
Methods
Staining of peptides

After Electrophoresis or Chromatography, the dried paper sheet is examined under ultraviolet light to locate fluorescent spots (oxidized Tryptophan residues). The Peptides are then visualized by Treatment with fluorescamine [74] and/or a ninhydrin–cadmium acetate reagent [28].

Staining with fluorescamine enables subsequent preparative elution of peptides from two-way paper maps and allows monitoring the accuracy of strip excision following one-dimensional Separation (usually by chromatography in BAW). Ninhydrin staining (of a side strip) is less sensitive than fluorescamine and sometimes fails to detect peptides with N-terminal Ile and Val residues, but it is more reliable because the color intensity is directly proportional to the peptide amount. Unlike fluorescamine, ninhydrin stains peptides with N-terminal Proline residues. Using this reagent, one can even differentiate N-terminal amino acid residues to a certain extent by the resulting spot color: red for Most Amino Acids, yellow for Gly and Ser, and orange gradually turning to red for Asn and Cys. The combined use of fluorescamine, ninhydrin, and specific Color Reactions for particular amino acids helps to precisely localize peptides in complex mixtures during one-dimensional separation.

10.8.3.1. Fluorescamine staining. Fluorescamine is stored as a solution in anhydrous acetone at a concentration of 0.1 mg/ml. Before use, 1 ml of this solution is added to 100 ml of 1% (v/v) pyridine in acetone. The paper is immersed in the solution and then dried in a stream of warm (or room-Temperature) air. If fluorescent spots do not appear after inspecting the chromatogram under an ultraviolet lamp, the Procedure is repeated with an increased fluorescamine concentration (2 ml of the stock solution per 100 ml of pyridine).

10.2.3.2. Ninhydrin–cadmium acetate staining. Ninhydrin is stored as a 0.25% (w/v) solution in acetone, and cadmium acetate is stored in aqueous acetic acid (1 g of Cd(CH₃CОО)₂∙2H₂О per 50 ml of glacial acetic acid and 100 ml of Water). The paper is immersed in a mixture prepared from 100 ml of the ninhydrin solution and 15 ml of the cadmium acetate solution, dried at room temperature, and then heated at 100 °C for 3 min. After color development, the chromatogram is left overnight in a fume hood for full color development and visualization of slow-reacting peptides.