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. 2007 Apr 26;404(Pt 1):131–140. doi: 10.1042/BJ20061747

Figure 4. Characterization of double point mutants of rat GDNF.

Figure 4

(A) Western blot of baculovirus-expressed mutants of GDNF. Conditioned supernatants from infected Sf9 cells were subjected to ion-exchange chromatography on SP-Trisacryl and GDNF immunoreactivity fractions were dialysed against PBS. The loadings shown were adjusted to give comparable intensities of development. Lane 1, commercial rat GDNF; lane 3, baculovirus-expressed wild-type GDNF; lane 5, K81A/K84A double point mutant; and, lane 7, R88A/R90A double point mutant. Lanes 2, 4 and 6 show similarly processed culture supernatants from mock-infected Sf9 cells, treated and grown in parallel to the cultures yielding the supernatant shown on the immediate right-hand side. (B) Heparin chromatography of wild-type (Wt) and double point mutants of GDNF. Samples of the immunoreactive SP-Trisacryl eluate fractions were dialysed against PBS and applied to 1 ml HiTrap Heparin columns. Fractions were assayed for GDNF immunoreactivity by heparin-binding ELISA (closed squares and solid line) and for conductivity (closed circles and dashed line).