[Production and application of rabbit polyclonal antibody against ARRDC5]

Objective To produce rabbit polyclonal antibody against arrestin domain-containing protein 5 (ARRDC5). Methods The recombinant prokaryotic plasmid pET-30a-Arrdc5 was constructed using molecular cloning techniques, introduced into E. coli BL21 (DE3), subsequently induced with isopropyl β-D-1-thiogalactopyranoside (IPTG) to express the recombinant ARRDC5 protein. After purification with Polyhistidine-tag (His-Tag) affinity column, New Zealand white rabbits were immunized with ARRDC5 protein. The antibody titer was assessed by ELISA. ARRDC5 protein expression in mouse testes was detected by Western blot. IP-MS was performed to identify the interacting proteins of ARRDC5 in the testis. Results The pET-30a-Arrdc5 recombinant plasmid was constructed correctly, and recombinant ARRDC5 protein was expressed and purified successfully by IPTG induction. After immunization, the titer of ARRDC5 antibody reached 1:512 000. The ARRDC5 protein was specifically recognized by ARRDC5 antibody in mouse testes. A total of 365 ARRDC5-interacting proteins were identified, which were significantly enriched in spermatogenesis-related biological processes. Co-IP results validated the interaction between ARRDC5 and pyruvate kinase isozyme M2 (PKM2). Conclusion The rabbit polyclonal antibody against ARRDC5 was generated successfully, which facilitates further investigation of ARRDC5 in spermatogenesis.