Classico western hrp substrate

The proteins were detected using the Millipore Luminata TM Crescendo and Classico
Western HRP Substrate. Each PVDF membrane received 1 ml of the HRP Substrate and allowed to incubate. The Western blot was imaged using a BioRad ChemiDoc gel imaging system. The program set-up was as follows: excitation was set to 480 nm and emission is 528 nm (using the Excitation Wheel 1 and Emission Wheel 1 respectively). The temperature was set to 37°C, and the plate was continuously shaken (orbital) for the duration of the experiment. The kinetic experiment lasted 16.5 hours, with reads of OD600 and fluorescence (taken from the bottom of the wells) every 30 mins, with the lid on the plate to prevent evaporation. Prior to the kinetic run, the plate was shaken for 15 seconds, following this an OD600 and fluorescence reading was taken for the time 0 points. The data was recorded by the Gen5 software, from which the data was exported in an excel file. The plate reader automatically subtracted the media control OD600 and fluorescence from all the experimental wells. The corrected fluorescence values were divided by the corrected OD600 values, and the technical and biological replicates were averaged to generate the final expression values for the esp promoter.