Goat anti mouse igg hrp conjugate

Expression vectors containing EGFP-tagged teneurin paralogues were co-transfected with tagRFP/Myc-tagged teneurin paralogues into Neuro2A cells with Lipofectamine 2000, the cell lysates collected after 2 DIV and immunoprecipitated with GFP-Trap agarose beads (ChromoTek, Germany). An EGFP-only vector was used as the bait control and co-transfected with the prey tagRFP/Myc-tagged teneurin paralogues. The precipitated products were separated using SDS-PAGE gel electrophoresis, transferred onto PVDF membrane and blocked for 1 h with 5% milk powder in TBS-Tween 20 (TBST) blocking solution before incubating overnight with primary antibody in blocking solution at 4°C. After primary incubation, blots were washed three times in TBST before incubation with secondary antibody in blocking solution for 2 h at RT. Blots were washed another three times in TBST before developed using Novex ECL chemiluminescent substrate reagent kit (Thermo Fisher Scientific, UK) and visualised on the Odyssey Fc imaging system (LI-COR Biosciences, UK). Blots were chemically stripped and re-probed with different antibodies. Antibodies were used at the following concentrations: Primaries mouse anti-myc tag (1:1,000; Cell Signaling) and chicken anti-GFP (1:5,000; Abcam), and secondaries goat anti-mouse IgG HRP conjugate (1:5,000; Abcam) and goat anti-chicken IgY HRP conjugate (1:5,000; Abcam).