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. Author manuscript; available in PMC: 2016 Apr 1.
Published in final edited form as: Insect Biochem Mol Biol. 2015 Feb 17;59:58–71. doi: 10.1016/j.ibmb.2015.02.005

Figure 5.

Figure 5

SDS-PAGE and immunoblot analysis of recombinant forms of MCO1. A) Purified recombinant forms of MCO1 were analyzed by SDS-PAGE using reducing (R) or non-reducing (NR) conditions followed by Coomassie staining. Each lane contains 1 μg protein. B) Purified recombinant forms of MCO1 were analyzed by SDS-PAGE using reducing conditions followed by immunodetection. Each lane contains 50 ng protein. DmMCO1T and AgMCO1T cleavage products that are linked by a disulfide bond under non-reducing conditions are indicated by an asterisk. A minor, uncharacterized cleavage product of DmMCO1 is indicated by a plus sign. Multimers of MsMCO1 that form after heating in reducing SDS-PAGE sample buffer are indicated by an arrow head. Note that the highest molecular mass multimers are not visible in panel B because proteins 200 kDa or greater did not transfer to the nitrocellulose membrane. The expected mass of each recombinant protein: DmMCO1T, 79.8 kDa, DmMCO1T amino-terminal cleavage product, 34.1 kDa; DmMCO1T carboxyl-terminal cleavage product, 45.7 kDa; DmMCO1T[R454A], 79.8 kDa; AgMCO1T, 81.3 kDa; AgMCO1T amino-terminal cleavage product, 35.3kDa (plus possible O-linked glycosylation, which may explain why this product co-migrates with the 46.1 kDa carboxyl-terminal product); AgMCO1T carboxyl-terminal cleavage product, 46.1 kDa; AgMCO1T[R542S], 81.3 kDa; TcMCO1Ts, 72.1 kDa; TcMCO1Tl, 75.3 kDa; MsMCO1Ts, 82.8 kDa; MsMCO1Tl, 86.0 kDa. (Abbreviations: DmT, DmMCO1T; DmT[RA], DmMCO1T[R454A]; AgT, AgMCO1T; AgT[RS], AgMCO1T[R542S]; TcTs, TcMCO1Ts; TcTl, TcMCO1Tl; MsTs, MsMCO1Ts; MsTl, MsMCO1Tl.)