Isoelectric focusing of oligopeptides: Detections by specific stains

By using ultrathin (350 μm) polyacrylamide gels, which at the end of the fractionation are pasted to filter paper and dried in an oven at 110°C, and after isoelectric focusing it has been possible to detect oligopeptides in the di- to tetradecapeptide range, which could not be detected by protein staining techniques. This is achieved by developing a series of specific stains for the following amino acid: Arg, Tyr, His, Trp, Met and Cys. Except for Met and Cys, the detection limits appear to be in the order of 0.2-2 μg of free amino acid loaded in the gel. The Pauli reaction for His and Tyr and the Sakaguchi stain for Arg can be developed sequentially in the same gel, thus allowing the detection of four different amino acids since, under these conditions, also Trp reacts. Unfortunately, more general reactions, such as the permanganage, the "Lowry" and the ninhydrin stains, cannot be utilized since the carrier ampholytes react very strongly with all these reagents.