*3.15. SDS-PAGE and Western Blotting*

Whole cell-lysate proteins and recombinant proteins were separated by electrophoresis on 12% PAG under the standard denaturing conditions according to the method of Laemmli [45] with prior heating of the sample (5 min at 100 ◦C) in a buffer containing 2% SDS, as well as under non-denaturing conditions, using the method of semi-native PAGE [46]. In the latter case, the samples were dissolved at 0 ◦C in a buffer containing 0.1% SDS, without β-mercaptoethanol, and separated on a gel without SDS at 10 mA for 2.5 h at 5 ◦C. A set of colored proteins (Fermentas, Lithuania) with molecular weights of 10, 15, 25, 35, 40, 55, 70, 100, 130, and 170 kDa were used as markers. The proteins separated in the gel were stained with Coomassie R-250 in 10% acetic acid and 30% methanol. GFP fluorescence after SDS-PAGE was recorded using the VersaDoc imaging system (Bio-Rad, USA). The determination of the recombinant proteins molecular weight was carried out from the graph of the linear relationship between log MW of marker proteins (from 15 to 70 kDa) and relative migration distance (Rf) of these proteins according to the formula y = −0.0147x + 2.0684 (R2 > 0.994).

The localization of PldA in PldA-GFP IBs was assessed by Western blotting. After SDS-PAGE, proteins were transferred from non-stained gel on nitrocellulose membrane (0.2 μm, Merk Millipore, Burlington, MA, USA) by equipment for semi-dry transfer at the current of 0.8 mA/cm<sup>2</sup> overnight at 4 ◦C according to the standard procedure [47]. Immunodetection was carried out by the protein detection system SNAP i.d. according to the instruction of the manufacturing company (Merk Millipore, Burlington, MA, USA). A murine polyclonal antiserum to recombinant PldA was prepared as described in [48]. HRP-Goat Anti-Mouse antibodies (Invitrogen, Waltham, MA, USA) were used according to the instruction of the manufacturing company. Antigen-antibody complexes were identified on nitrocellulose membrane by the hydrogen peroxide detection with 3,3- -diaminobenzidine for 20 min at the room temperature.
