Fatin Safiudin The discrepancy between your chromatogram, PAGE gel, and plate assay results likely arises because the absorbance at 280 nm and PAGE gel primarily reflect total and abundant protein content, while the fluorescence-based plate assay detects functional activity or fluorescent compounds, which can be present in much lower amounts. The strong fluorescence observed in E3, E6, E7, and E9, despite the absence of corresponding protein bands on the gel, suggests these fractions may contain either low-abundance active protein, degraded fluorescent fragments, or even non-protein fluorescent contaminants that are below the detection limit of Coomassie staining. Additionally, E2 may contain aggregated or misfolded protein that contributes to the A280 peak and visible band on gel but has reduced or no functional fluorescence. Fluorescence is a highly sensitive readout and can detect even trace amounts of active or tagged molecules, explaining why activity is seen in fractions where no clear protein bands are visible.