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. 2017 Apr 21;15(4):123. doi: 10.3390/md15040123

Figure 2.

Figure 2

Interference levels caused by crude, heat- and TCA-clarified reference extracts on the enzymatic assays. (A) Intrinsic proteolytic activity of crude and clarified extracts (final dilution 1/2) on the immobilized peptidic substrate DU2 under Plm II enzymatic assay conditions (2 h at 37 °C in buffer 100 mM NaAc, pH 4.7). The proposed threshold for substrate degradation of 30% is indicated by a dashed line. (B) Intrinsic proteolytic activity of crude and clarified extracts (final dilution 1/20) on the fluorogenic substrate Z-FR-AMC (12.5 μM) under FP2 enzymatic assay conditions (buffer 100 mM NaAc, 10 mM DTT pH 5.5 buffer). The proposed threshold for intrinsic substrate degradation (∂F/∂t ≥ 5 × 10−4 AFU·s−1) is indicated by a dashed line. (C) Effects of crude and clarified extracts (final dilution 1/20) on fluorescence readouts (λexc/λemss = 355 nm/460 nm) of the AMC standard. The limit for non-significant quenching effect (Q = FAMC/FAMC + EXT = 1) is indicated by a dashed line. The experiments were all performed in triplicate.