Figure 5

[α-sec-EAP] and [unselected proteins,UP] secreted by E(M) are >50 and upto 4 fold higher than those secreted by E(P). (Figure 5a) Incubation of the panel of cultures, shown in Figure 3 (with the exception of E(M9)-B, in lane 6) was for 170 hours with supplementation of methanol inducer at 24 hour intervals. Culture supernatants were harvested as determine in the materials and methods. Aliquots (20 μL) of cultures of E(P), h-V, H, E(M9)-B, EM32)-A,-B,-C,-D and E(M44)-A,-B were loaded in the order shown above the lanes (lanes 3–12) and co-electrophoresed with broad range markers (lane 1) and EAP purified from E.coli (0.08 units/lane, lanes2) through denaturing (4%-20%) polyacrylamide gradient gels with Laemmli electrophoresis buffer. The gel was stained with Gel Code Blue (GCB) scanned and saved as TIFF files. The respective ROIs of proteins corresponding to EAP, α-sec-EAP, unselected protein-1 (UP1) and unselected protein 3 (UP3) are identified and color coded on the left and right of the gel. The ROIs corresponding to the above 4 proteins were densitometrically scanned and normalized to UP3 in lane 3 and plotted as their respective ratios to E(P). Normalized ROIs corresponding to EAP, α-sec-EAP and HSA in E(P), v-H, H and E(M) were plotted with Excel programs (Figure 5b). Normalized ROIs corresponding to UP1 and UP3 were plotted as their ratios in EAP/E(P), E(P)/E(P), v-H/E(P), H/E(P) and all E(M)/E(P) (Figure 5c, 5d).