Alexafluor555 conjugated goat anti mouse igg2a

To generate an NT2-AnkG-GFP expression construct, we cloned Ank3-exon1e cDNA (Open Biosystems, catalogue #MMM1013-64925) into pEGFP-N1. Vann Bennett provided a rat NT3-AnkG270-GFP plasmid and affinity-purified rabbit anti-All AnkG antibodies (raised against a large C-terminus fusion protein). Isoform-specific rabbit antibodies against portions of the NT1-AnkG, NT2-AnkG and NT3-AnkG peptides were raised (YenZym, Inc., S. San Francisco, CA) and affinity-purified using methods described previously(22 (link)). Antigenic peptides used were derived from the human sequences, with a C-terminal cysteine residue added for conjugation: NT1 (NT1-AnkG residues 8–29): SPAGTEDSAPAQGGFGSDYSRSSRKSC; NT2 (residues 2–21): SEEPKEKPAKPAHRKRKGKKC; NT3 (residues 2–21): AHAASQLKKNRDLEINAEEEC. Purchased primary antibodies were mouse N-terminal isoform non-selective, called here “All AnkG” (IgG2a, clone N106/36; UC Davis/NIH NeuroMab Facility; Davis, CA), mouse anti-PV (IgG1, clone Parv-19, Sigma-Aldrich; St. Louis, MO), and mouse anti-PanNaV (IgG1, Sigma-Aldrich). Corresponding secondary antibodies were dylight488 donkey-anti-rabbit IgG (Jackson ImmunoResearch; West Grove, PA), AlexaFluor647-conjugated goat anti-mouse IgG1 (Invitrogen; Eugene, OR), AlexaFluor555-conjugated goat anti-mouse IgG2a (Invitrogen), and, for western blots, HRP-conjugated anti-rabbit IgG (Jackson).