Anti mouse igg peroxidase polyclonal goat antibody

Proteins were separated by NuPAGE 4–12% Bis-Tris gels (ThermoFisher Scientific) and transferred to nitrocellulose membranes (Amersham Protran Premium, 0.45 μm). The membranes were blocked with 5% nonfat dry milk (Sema5A_TSR3-4 proteins with biotinylated Avitag) or 3% Bovine Serum Albumin (His-tagged Sema5A_sema-TSR1-7 proteins) in PBS for 1 h at room temperature. For His₆ tag detection, the membranes were incubated with primary antibody (6xHis Monoclonal Antibody, TaKaRa, cat. no. 631212 dilution 1:3000) for 1 h at room temperature. Blots were then washed six times for 5 min with PBS-0.1% Tween-20 and incubated for 1 h at room temperature with secondary antibody conjugated to horseradish peroxidase (Anti-mouse IgG peroxidase polyclonal goat antibody, Sigma, cat. no. A0168, dilution 1:10,000). For biotinylated Avitag detection, the blot was incubated with Streptactin HRP (BioRad) antibody at 1:25,000 dilution for 1 h at room temperature. This was followed by washing of blots six times for 5 min with PBS-0.1% Tween-20, and signal detection using ECL (BioRad).

Proteins were separated by NuPAGE 4–12% Bis-Tris gels (ThermoFisher Scientific) and transferred to nitrocellulose membranes (Amersham Protran Premium, 0.45 µm). The membranes were blocked with 5% nonfat dry milk (His6 tagged proteins) or 5% BSA (TwinStrep tagged proteins) in PBS for 3 h at room temperature. For His6 tag detection, the membranes were incubated with primary antibody (Penta-His Antibody, Qiagen, cat. no. 34660, dilution 1:1000) for 1 h at room temperature, washed three times for 10 min with PBS and incubated for 1 h at room temperature with secondary antibody conjugated to horseradish peroxidase (Anti-mouse IgG peroxidase polyclonal goat antibody, Sigma, cat. no. A0168, dilution 1:10,000). For TwinStrep tag detection, the membranes were incubated with antibody conjugated to horseradish peroxidase (Precision Protein StrepTactin-HRP Conjugate, BioRad, cat. no. 1610380, dilution 1:1000) for 1 h at room temperature. After washing three times for 10 min with PBS, the signal was detected using ECL (BioRad). Uncropped images of all western blots are shown in the Source data file.