Hrp conjugated goat anti rabbit secondary antibody

SDS-PAGE was performed as described previously [51] using 12% (w/v) polyacrylamide slab gels. For western blot analysis, the protein samples were electroblotted onto polyvinylidene difluoride membranes using a Trans-Blot cell (Bio-Rad). After transfer, the membranes were probed with the appropriate primary antibodies and HRP-conjugated goat anti-rabbit secondary antibody (Promega), and the signals were detected using an ECL kit (GE, Evansville, IN, USA). The primary antibodies were diluted as follows: polyclonal antibody against MAPK6 (1∶1,000), HSP18.2 (1∶2,000), NCED (1∶3,000), dehydrin (1∶3,000), and actin (1:2,000).

SDS-PAGE was performed as described previously [54] (link) using 12% (w/v) polyacrylamide slab gels. For Western blot analysis, the protein samples were electroblotted onto polyvinylidene difluoride (PVDF) membranes using a Trans-Blot cell (Bio-Rad). After transfer, the membranes were probed with the appropriate primary antibodies and HRP-conjugated goat anti-rabbit secondary antibody (Promega, Madison, WI, USA) and the signals were detected using an ECL kit (GE Company, Indianapolis, IN, USA). The primary antibodies (all obtained from Agrisera, Inc., Vannas, Sweden) were diluted as follows: polyclonal antibody against plant 9-cis-epoxycarotenoid dioxygenase (NCED; 1∶3000), dehydrin (1∶3000), heat shock protein 18.2 (HSP18.2; 1∶2000), mitogen-activated protein kinase 6 (MAPK6; 1∶1000), and actin (1∶2000).

Oocytes expressing full-length receptors or different combinations of the split-intein receptor fragment fusion proteins were isolated 3–4 days after RNA injection and washed twice with phosphate-buffered saline (PBS). Total cell lysates were obtained by lysing the oocytes in Pierce™ IP lysis buffer with added Halt protease inhibitor cocktail (Thermo Fisher Scientific). Surface proteins were purified with the Pierce™ Cell Surface Protein Isolation Kit (Thermo Fisher Scientific). Purified surface proteins or total cell lysates were run on a 4–12% BIS-TRIS gel (for P2X2) or 3–8% Tris-acetate gel (for Na_V1.5) and transferred to a polyvinylidene difluoride membrane. Membranes were incubated with rabbit polyclonal anti-Na_V1.5 (#ASC-005, Alomone labs; 1:2000), anti-Na_V1.5 (#ASC-013, Alomone labs; 1:1500) or anti-P2X2 Antibody (#APR-003, Alomone labs; 1:2000) and the bound primary antibodies were detected by a HRP-conjugated goat anti-rabbit secondary antibody (W401B, Promega; 1:2000). Membranes were developed and visualized using the Pierce™ ECL immunoblotting substrate (Thermo Fisher Scientific).