**Jess Simple Western**

JESS Simple Western™ instrument (ProteinSimple®, Bio-Techne, Minneapolis, MN, USA) is a fully automated and capillary-based system that performs all downstream steps of sample preparation [46]. For preparation of the separation module a 2– 40 kDa separation module from ProteinSimple was used which includes a capillary cartridge, pre-filled microplates, wash buffer, 10 sample buffer, lyophilized fluorescent 5x master mix, lyophilized DTT and a lyophilized biotinylated ladder. Standard

pack reagents were prepared per manufacturer recommendations, for the 400 mM DTT solution preparation, the fluorescent 5X master mix and the ladder. The samples and *h*EGF standard were prepared. The samples and the standard *h*EGF, with a known concentration, were serially diluted down to 3–104 ng/mL using 0.1 sample buffer. Then, fluorescent 5X master mix was added to all measured samples, so the final mixture is 1 part master mix and 4 parts sample. The samples and standards were heated at 95°C for 5 minutes. The samples were then mixed again by vortex and then spun down and stored on ice. For detection, an anti-rabbit detection module from ProteinSimple was used which includes anti-rabbit secondary antibody, Luminol S, Peroxide, Streptavidin-HRP and antibody diluent. For preparation of the primary antibody, polyclonal rabbit anti-*h*EGF antibody (ab9695, Abcam) was diluted in 1:50 ratio with antibody diluent. For the preparation of the chemiluminescence substrate, 200 μL of luminol-S and 200 μL of peroxide was mixed in a microcentrifuge tube. Then the prepared samples, the biotinylated ladder, antibody diluent for washing and blocking, the diluted primary antibody, the secondary antibody, Streptavidin-HRP for biotinylated ladder capillary only, and luminol-peroxide mix were pipetted into the microplate. The plate was spun down at 1000 g for 5 minutes followed by addition of wash buffer and all air bubbles removed with ethanol vapor and the plate placed into Jess and the right program selected in Compass Simple Western ™ software.
