Sodium cacodylate buffer

The samples before and/or after cell cultivation were washed in PBS, fixed at RT in a glutaraldehyde containing sodium cacodylate buffer (Carl Roth, Karlsruhe, Germany) and stored overnight at 4°C. SEM preparation was performed as described previously [29 (link), 30 (link)]. Then, the samples were dehydrated in increasing alcohol concentration (from 10 to 100%) and dried in hexamethyldisilazane (HMDS, Sigma-Aldrich, Taufkirchen, Germany) as described previously [31 (link)]. Finally, all samples were coated with carbon, and studied in field emission scanning electron microscope Phillips FESEM XL30 (FEI, Eindhoven, Netherlands) at 5 kV in secondary electron (SE)-mode and at 10 kV accelerating voltage (10 mm working distances) in backscattered electron (BSE)-mode.

The purified protein sample was estimated to be 90% pure, based on SDS-PAGE gels. Tris buffer, sodium cacodylate buffer, LB broth, kanamycin, isopropyl-ß-D-thiogalacto-pyranoside (IPTG), imidazole, sodium dodecylsulphate (SDS), NaCl, acrylamide/bisacrylamide solution 29:1, (30% w/v), N,N,N′,N′-tetramethylethylenediamine (TEMED), ammonium peroxide, Rotigarose His/Ni beads, methanol, glycerol, glycine, sodium chloride, powdered milk, and acetic acid were acquired from Carl Roth (Karlsruhe, Germany). Amonium-acetate buffer, ammonium persulphate (APS), β-mercaptoethanol, Tween 20, bromphenol blue, MOPS buffer, and diphenylamine (DPA) were acquired from Sigma-Aldrich (St. Louis, MO, USA). The Arg₂-2-naphthylamide (Arg-Arg–2NA) was produced by Bachem (Bubendorf, Switzerland); 2-naphthylamine (2-NA), 2-mercaptoethanol, and DNase I from bovine pancreas were produced by Merck (Darmstadt, Germany). For visualization of SDS-PAGE gels, we used PhastGel Blue R tablets (Pharmacia, Uppsala, Sweden). The EDTA was obtained from Kemika (Zagreb, Croatia), and metal (zinc, copper, cobalt, manganese) standard nitrate solutions were produced by Merck (Darmstadt, Germany).