2.2 Recombinant ZnT8/GAD65 expression and purification from cell
cultures
For the ZnT8/GAD65 expression, independent Sf9 (serial passage 30)
suspension cultures
in log-phase at a cell density of 4 x 107 cells in 30
mL (2 x 106 cell/mL) were infected with
Acpolh‐ZnT8/GAD65 MOI 1. After 4 days of incubation in the dark at 27
°C, in continuous agitation, cells were centrifuged. The recombinant
chimera was recovered following the protocols of lysis and purification
previously described by our group (Trabucchi et al., 2019).
For the determination of total protein concentration Bradford microassay
(Bradford, 1976) was performed using Coomassie Plus™ Protein Assay
(Thermo Fisher Scientific).
The recombinant ZnT8/GAD65 chimera was stored with a mixture of 50% v/v
glycerol, Tween 20 and protease inhibitors, and kept at -20°C until
used.
2.3 Sodium dodecyl sulphate-polyacrylamide gel
electrophoresis and western blot analysis
The protocols followed for SDS-PAGE and WB analysis were the same as
Faccinetti et al. (2017) and Trabucchi et al. (2019). Briefly, protein
fractions were separated by 10% SDS-PAGE under reducing conditions,
followed by Coomassie Brilliant Blue R-250 staining. For detection by
WB, once the protein bands were transferred onto nitrocellulose
membranes, the expression of the chimera was evidenced by either a mouse
monoclonal antibody to GAD65, a rabbit policlonal antibody to ZnT8 or a
mouse monoclonal anti-histidine antibody (BD Pharmingen). Bound
antibodies were visualized by incubation with peroxidase-conjugated goat
antibodies to mouse IgG or with peroxidase-conjugated goat antibodies to
rabbit IgG, respectively. Protein bands were visualized by the addition
of α-chloronaphthol (Sigma-Aldrich) and 10 vol.
H2O2.