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. Author manuscript; available in PMC: 2013 Apr 26.
Published in final edited form as: Protein Expr Purif. 2011 Oct 19;81(2):175–180. doi: 10.1016/j.pep.2011.10.006

Figure 2. Recombinant κGalNAc-T2 protein is secreted by Sf9 cells and 293T cells.

Figure 2

Recombinant protein κGalNAc-T2 was expressed in human HEK 293T cells and in insect Sf9 cells. Cell-culture medium supernatants (S) and cell lysates (C) were analyzed by SDS-PAGE followed by western blotting with anti-His tag HRP-conjugated antibody. For comparison, native GalNAc-T2 (TM) containing transmembrane domain was expressed in 293T cells and analyzed in parallel. κGalNAc-T2 was secreted by both cell types, whereas the full length, transmembrane domain-containing form remained in 293T cells as expected. Predicted molecular mass of κGalNAc-T2 is 65.8 kDa and that of GalNAc-T2 is 64.7 kDa. For analyses of GalNAc-T2 from insect cells (Sf9), 50-µl aliquots of cell suspension were centrifuged and the supernatant and cell pellet were separated and mixed with SDS-PAGE sample buffer. For analysis of GalNAc-T2 from adherent cells, cell-culture supernatant was removed, adherent cells were trypsinized and then resuspended in the original volume of fresh culture medium. Equal aliquots of supernatant and trypsinized cells were mixed with SDS-PAGE sample buffer and equal volumes were used for western blot analysis.