The generation of polyclonal rabbit antiserum against prokaryotic-expressed rgpTNF-α has been described earlier [18 (link)]. In brief, the rgpTNF-α was mixed with the adjuvant TiterMax Gold (CytRx Corp, Norcross, GA) and injected subcutaneously into New Zealand white rabbits in four injections that were spaced at 3-week intervals. The animals were exsanguinated 5 weeks following the last booster and sera were collected by centrifugation of blood at 1500 rcf for 20 minutes. The serum was aliquoted and stored at −80°C until it was used in the Western blot assay.
Approximately 200 ng of eukaryotic-expressed rgpTNF-α protein was run on a 10–20% tricine gel (Invitrogen) and blotted onto a nitrocellulose membrane using the semidry Minitrans blot electrophoretic transfer cell apparatus (Bio-Rad). The identity of the eukaryotic-expressed rgpTNF-α was determined by its reaction to the polyclonal anti-rgpTNF-α antiserum by Western blot analysis using the WesternBreeze chromogenic kit (Invitrogen) following the manufacturer's instructions with anti-rabbit (IgG) antibody as the secondary antibody (Invitrogen).
Approximately 200 ng of eukaryotic-expressed rgpTNF-α protein was run on a 10–20% tricine gel (Invitrogen) and blotted onto a nitrocellulose membrane using the semidry Minitrans blot electrophoretic transfer cell apparatus (Bio-Rad). The identity of the eukaryotic-expressed rgpTNF-α was determined by its reaction to the polyclonal anti-rgpTNF-α antiserum by Western blot analysis using the WesternBreeze chromogenic kit (Invitrogen) following the manufacturer's instructions with anti-rabbit (IgG) antibody as the secondary antibody (Invitrogen).