Protein concentration was measured by the Bradford method [55], using BSA as the standard. The fraction containing mono-PEG-StAP3 species was the employed for biological studies. Prior to assays, this fraction was dialyzed against 20 mM Tris–HCl pH 8, for 48 h at 4 °C, using a cellulose membrane (Sigma D9652-100) to remove DTT and SDS. selleck chemicals The fraction was then stored at −20 °C for further analyses. To evaluate the effect of mono-PEG-StAP3 on the germination of F. solani spores, in vitro bioassays were performed as described by Guevara et al. [26]. To quantify the effect of mono-PEG-StAP3 on spore germination, the bioassays were examined by observation of four fields in Neubauer camera with a bright-field microscope. The results

from three independent experiments were analyzed to calculate the percentage of inhibition. B. cereus and E. coli were grown in Luria–Bertani selleck medium at 37 °C with continuous shaking to exponential phase. The bacteria were harvested from broth by centrifugation at 3500 rpm for 10 min, washed and resuspended in sterile PBS at a concentration of 104 c.f.u./ml. The concentration of bacteria was verified and quantified by culture on sheep blood agar plates. One hundred microliters of bacterial suspension were plated on 96-well polystyrene microtiter plates (BD Biosciences), and serial dilutions of mono-PEG-StAP3 were added to individual wells in triplicate and incubated for 6 h at 37 °C

with rocking. Bacteria were subsequently dispersed and aliquots were plated on blood agar plates to obtain colony counts. Pathogen viability after protein treatment was determined from the number of colonies obtained on the buffer-treated control plates compared to the number of colonies from protein-treated samples. The half maximal inhibitory concentration

(IC50) was calculated as the concentration of protein required to inhibit microbial growth by 50%. F. solani spores were incubated overnight at 25 °C with water as control or exposed to different 3-oxoacyl-(acyl-carrier-protein) reductase amounts of mono-PEG-StAP3, as described by Guevara et al. [26]. SYTOX Green probe (Molecular Probes) was added to a final concentration of 0.5 μM and qualitative detection of SYTOX Green uptake was performed. After 30 min incubation, the fluorescence of the sample was observed with a Nikon Eclipse E200 fluorescence microscope (Nikon, Tokyo, Japan) equipped with a B-2A Fluorescein filter set. Positive controls included spores treated with 0.5% (w/w) Triton X-100. Fluorescence was measured using a FluorosKan Ascent (Thermo Electron Corporation, Finland) fluorescence measurement system at an excitation wavelength of 480 nm and an emission wavelength of 530 nm. Fluorescence values were corrected by subtracting the fluorescence value of a buffer incubated with SYTOX Green. Fresh human red blood cells (hRBC) were rinsed in PBS, centrifuged for 10 min at 800 rpm three times, and resuspended in PBS to a final erythrocyte concentration of 4% (v/v).