As an alternative to the multi-alanine scanning strategy, random substitutions could be also used to explore the effect of side chains with different functional groups. Similarly to alanine scanning, the method is based on a systematic substitution of each residue, however the substitutions are selected randomly. Those substitutions can be selected from all the 20 natural L-amino acids set (Fig. ), or from reduced set representative of the natural L-amino acids (Fig. ). In this work, the reduced set is composed of the following amino acids: His, Arg, Glu, Trp, Val, Ser, Asn, Cys, Pro and Tyr. In the case of the random scanning by properties, each position is randomly substituted by one amino acid of each amino acid property class (Fig. ). This ensures that all the amino acid properties are represented in the final mutated sequences. For this study, the amino acids were gathered into the following property classes: polar positive (Arg, His, Lys), polar negative (Glu and Asp), polar neutral (Gln, Thr, Gly, Cys, Asn and Ser), non-polar (Ile, Ala, Leu, Pro, Val and Met) and non-polar aromatic (Tyr, Trp and Phe). Depending on the sequence length, multiple representations of the same class can be present for some positions to meet the desired total number of mutated sequences (i.e. 96 for a 96 well-plate).