Protein a g agarose kit

Immunoprecipitation was performed using a protein A + G agarose kit (# P2012; Beyotime) according to the manufacturer’s instructions. Briefly, cells were lysed with radioimmunoprecipitation assay (RIPA) buffer (#P0013B; Beyotime). The prepared lysates were immunoprecipitated with anti-CypD antibodies (Bioworld Technology, Inc., Nanjing, China) at 4 °C for 12 h. Each experiment using this assay was performed at least three times independently. Immunoblotting was performed with the following primary antibodies: anti-Ac-Lys antibody, or anti-mouse V5 Tag monoclonal antibodies. A goat anti-mouse horseradish peroxidase-conjugated antibody was used as the secondary antibody.

The recombinant IgM (AGR34023.1), IgD (AGR34025.1), IgZ (AGR34024.1) and CD8 (XP_048023282.1) protein were prepared in the same way as OmpA. After three immunizations with purified recombinant protein, rabbit antiserum against IgM, IgZ, IgD, and CD8 were obtained and purified using Protein A/G Agarose Kit (Beyotime, Shanghai, China). The total proteins of M. amblycephala tissues were extracted with RIPA lysate (Beyotime) and the concentration was determined with a BCA kit (Beyotime).
The specificity of prepared polyclonal antibodies was verified with purified recombinant proteins and total tissue proteins of M. amblycephala by western blotting (28 (link)). Specifically, protein samples were separated on a 12% SDS-PAGE gel and transferred onto polyvinylidene difluoride (PVDF) membranes using a Trans-Blot apparatus (BioRad, Berkeley, CA, USA). Non-specific reactivity was blocked with 5% (w/v) skim milk powder in TBS (150 mM NaCl, 20 mM Tris-base, pH 7.4). The PVDF membrane was incubated with polyclonal antibodies (1: 2000) and HRP-conjugated goat anti-rabbit IgG (H+L) (Beyotime; 1: 2000) for 1 h, respectively. Finally, a DAB Kit (Beyotime) was used for detection.