Soluble protein expression using different solubility tags is protein dependent. Three proteins encoded by genes A (~48 kD), B (~49 kD) and C (~59 kD) with solubility problems were expressed in E.coli expression vectors (pD441-XXX) with various N-terminal solubility tags MBP (~42 kD), GST (~26 kD), PpiBT (~19 kD), Fh8 (~8.8 kD) and 6XHis as a negative control (~1 kD) under control of the IPTG-inducible T5 promoter, at 37°C and room temperature (RT). Higher soluble protein expression was observed at RT except for gene A with MBP tag. BL21 cells were transformed and grown at 37°C in 2 ml LB + 30 µg/ml kanamycin to an OD600 of 0.8. Cultures were induced by addition of 1mM IPTG and incubated for 3 hrs at 37°C or overnight at room temperature (RT); uninduced cultures were run as negative controls. Samples were pelleted, lysed and total and soluble preps prepared. Total protein (T) samples and soluble fractions (S) were denatured and reduced in sample buffer with reducing agent at 95°C for 10min. 5µl (approx. 5 O.Ds) of denatured sample was loaded per lane of a 4-12% NuPAGE Bis-Tris MES gel and Coomasie stained.  Band volumes (shown in red boxes) were measured and quantitated using BSA as a reference.