Freezone 18 liter console freeze dry system

O/N E. coli cultures, harvested from 2 l culture, were pelleted by centrifugation at 8000 × g for 15 min., resuspended in sonication buffer (50 mM Tris-Cl, 500 mM NaCl 20 mM imidazole) and disrupted by sonication (90 sec. for 6 times at 60% intensity) using a Vibra Cell sonicator (Sonics). Recombinant AtHSP70 protein was purified by affinity chromatography using the AKTA FPLC system (GE Healthcare, Tampa, FL, USA). In details, total proteins were loaded onto a His-Prep FF16/10 (GE Healthcare) column and washed with elution buffer with increased imidazole concentration (0–500 mM). r-AtHSP70 was eluted at 250 mM imidazole concentration. The recombinant protein was then dialyzed against TrisCl 50 mM pH 7.5 buffer and lyophilized (FreeZone 18 Liter Console Freeze Dry System; LabConco, Kansas City, MO, USA). The purity of r-AtHSP70 was checked by SDS-PAGE and Western blot.

Six randomly collected eggs from each replicate after 30, 60, 90, and 120 days of the experiment in all the examined groups were analyzed. Eggs were broken in order to separate albumen, yolk and shell. Egg yolks from each replicate (n = 6) were mixed homogeneously in order to obtain representative samples. Then, samples were kept in the temperature of −40 °C. In the next step, the samples underwent lyophilization using Free Zone 18 Liter Console Freeze Dry System (Labconco Corporation, Kansas City, MO, USA).

The yeast strain Candida albicans CCY 29-3-100 (serotype A) (CCY Culture Collection of Yeasts, Institute of Chemistry, Center for Glycomics, Slovak Academy of Sciences, Bratislava, Slovakia) was used to isolate and purify cellular mannan from fresh biomass. Mannan was extracted by autoclaving in 0.2 mol/l NaCl (120°C, 700 kPa) for 10 min and purified using precipitation with Fehling reagent according to a previously described method (Peat et al., 1961 (link)).
For the preparation of FITC-labeled mannan, C. albicans CCY 29-3-100 mannan (54 mg) was suspended in 1.00 mL of dimethyl sulfoxide and 2.0 μL of pyridine was added. The suspension was heated in a thermoblock at 95°C until the mannan dissolved (3 h). Then, 20 mg of isothiocyanatofluorescein (FITC) was added and heated for another 2 h at 95°C. The reaction was quenched by addition of 10 mL of water, and the result was dialyzed using cellulose membrane tube (cut-off = 14,000, Sigma) against 0.05 % NaHCO3 (1 × 0.9 L, 4 h stirred) and deionized water [8 × 0.9 L, 4 h on stirrer or 12 h in the refrigerator (5°C)] and then lyophilized (FreeZone 18 Liter Console Freeze Dry System, Labconco Corporation, Kansas City, USA).