Quantitative Analysis of Recombinant mCherry in E. coli

The fluorescent signal of mCherry produced in recombinant E. coli was measured with the Lumina fluorescence spectrometer (Thermo, USA) as previously described [22 (link)]. Fluorescence emission was recorded at 610 nm for mCherry, and the fluorescence value was normalized by dividing the fluorescence intensity by the OD₆₀₀ value of the same sample. A dot blot using cell lysate was performed as previously described [29 (link)]. Briefly, twenty microliters of induced culture was mixed with an equal volume of assay solution (200 mM Tris-HCl, pH 6.8, 2% SDS, and 200 mM DTT), and boiled for 10 min. An aliquot of 5 μL of each sample was dripped onto PVDF membrane. After the membrane was blocked and washed, it was incubated with primary mouse anti-His IgG (Tiangen, Beijing, China). After extensive washing, the membrane was then incubated with a secondary HRP labeled rabbit anti-mouse IgG (Sangon Biotech, Shanghai, China). Finally, the associated antibodies were visualized using an enhanced HRP-DAB chromogenic substrate kit (Tiangen, Beijing, China).