(A) Catalysis of RAL to RA by RALDHs/ALDH2, coupled with the generation of NADH from the cofactor NAD+, emitting fluorescence signals. (B) Three different buffer composition without Mg2+ (RB1, 2) and with Mg2+ (RB3). HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; and MPD, 2-methyl-2,4-pentanediol. (C–F) Effects of buffers RB1–3 and the absence or presence of His-tag on initial velocity (V0) of reactions for RALDH1 (C), RALDH2 (D), RALDH3 (E) and ALDH2 (F). Blue, yellow and gray colored graphs represent RB1–3 buffers, respectively. Error bars represent standard deviation of mean at 95% confidence (n=3). Statistical analysis including analysis of variance followed by Tukey’s post-hoc test was performed separately for each protein under two tag conditions (His-tagged, untagged) and three buffer conditions. Means that do not share a letter are significantly different (P<0.05).