Fig 1: Preparation of immunogen His-Mb protein and generation of plasma-derived rabbit anti-Mb polyclonal antibodies. (A) The alignment of Mb protein amino acids sequence in different species (Rattus, Mus, Homo, Equus, Sus, Bos, Ovis, Canis, Oryctolagus, Macaca, Gallus, and Danio). (B) Double enzyme digestion identification of recombinant pTT5-Mb plasmid by EcoRI and BamHI. Lane 1: pTT5-Mb plasmid; Lane 2: M is DNA ladder (O’GeneRuler DNA Ladder Mix 100–10,000 bp). (C) SDS-PAGE and Coomassie brilliant blue staining analysis of purified His-Mb protein. Lane 1: M represents protein marker (14.4–116 kDa); Lane 2: The supernatant of HEK-293F cells containing His-Mb protein passes through the Ni-NTA affinity chromatography column; Lane 3–4: Purified His-Mb protein. (D) Immunoprecipitation assays verified the His-Mb protein. Lane 1: M represents protein marker (10–250 kDa); Lane 2: Recombinant rabbit IgG (Isotype Control); Lane 3: Anti-His antibody. (E) Mass spectrometry analysis of purified His-Mb protein. (F) The comparison of antibody titers in three rabbits (Rabbit A, B, C) immunized with His-Mb protein that evaluated by indirect ELISA. Red, blue, and orange represent positive antiserum that contains plasma-derived anti-Mb polyclonal antibodies. Grey represents pre-immune negative serum. Antiserum was serially diluted fourfold from 1:250 to 1:256,000. (G–I) WB analysis for species reactivity of plasma-derived anti-Mb polyclonal antibodies of Rabbit A, Rabbit B, and Rabbit C. Lane 1: M is protein marker (11–245 kDa); Lane 2: Pure native horse Mb (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China, #M8090); Lane 3: Mouse muscle lysates (skeletal muscle lysis mixture of mice); Lane 4: Rat muscle lysates (skeletal muscle lysis mixture of rats); Lane 5: Pure native human Mb (Abcam, #ab96036); Lane 6: Pure recombinant human His-Mb (OkayBio, #K2916); Lane 7: Pure recombinant rat His-Mb protein (immunogen). The protein amounts of pure native horse Mb, mouse skeletal muscle lysate (mixture), rat skeletal muscle lysate (mixture), pure native human Mb, pure recombinant human Mb, and immunogen (rat His-Mb) are 5 µg (pure protein), 50 µg (total protein), 50 µg (total protein), 0.66 µg (pure protein), 0.264 µg (pure protein), and 0.21 µg (pure protein), respectively.
Fig 2: Validation of the species reactivity and tissue specificity of recombinant rabbit anti-Mb monoclonal antibody of transfection mass ratio of heavy chain and light chain is 1:2. (A) WB analysis for species reactivity of recombinant rabbit anti-Mb monoclonal antibody (transfection mass ratio of heavy chain and light chain = 1:2). Lane 1: M is protein marker (11–245 kDa); Lane 2: Pure native horse Mb (Beijing Solarbio Science & Technology Co., Ltd., #M8090); Lane 3: Mouse muscle lysates (skeletal muscle lysis mixture of mice); Lane 4: Rat muscle lysates (skeletal muscle lysis mixture of rats); Lane 5: Pure native human Mb (Abcam, #ab96036); Lane 6: Pure recombinant human His-Mb (OkayBio, #K2916); Lane 7: Pure recombinant rat His-Mb protein (immunogen). The protein amounts of pure native horse Mb, mouse skeletal muscle lysate (mixture), rat skeletal muscle lysate (mixture), pure native human Mb, pure recombinant human Mb, and immunogen (rat His-Mb) are 5 µg (pure protein), 50 µg (total protein), 50 µg (total protein), 0.66 µg (pure protein), 0.264 µg (pure protein), and 0.21 µg (pure protein), respectively. (B,C) The Mb protein expression in skeletal muscle, myocardium, liver, spleen, lung, and kidney of normal rats or mice was detected by WB using recombinant rabbit anti-Mb monoclonal antibody. Immunogen His-Mb protein acted as a positive control. (D,E) Immunoprecipitation of the recombinant rabbit anti-Mb monoclonal antibody with rat or mouse skeletal muscle lysates. IgG antibody acted as a negative control.
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