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. 2005 Nov 7;102(46):16584–16589. doi: 10.1073/pnas.0508306102

Fig. 1.

Fig. 1.

Purification of the LGN/Gαi-1/NuMA (LGN BD) complex. (A) A soluble Sf9 cell lysate containing rat His-6-LGN was supplemented with purified myristoylated Gαi-1 from E. coli and rapidly adsorbed to Ni-NTA agarose. The resin was eluted with imidazole, and a portion of the eluate was resolved by SDS/PAGE; the gel was stained with Coomassie brilliant blue. (B) The eluate containing LGN/Gαi-1 was immediately supplemented with a 5-fold molar excess (to LGN) of myristoylated Gαi-1, loaded onto a Hi-trap Q column (GE Healthcare), and resolved with a linear NaCl gradient. Fractions of the eluate were resolved by SDS/PAGE. The fractions that were pooled are noted (Q pool). (C) The Q pool of LGN/Gαi-1 complex was incubated with a 4-fold molar excess of NuMA (LGN BD) and a molar equivalent of myristoylated-Gαi-1, ultracentrifuged, and gel-filtered over tandem Superdex 75/200 columns. Fractions of the gel filtration eluate were resolved by SDS/PAGE and stained. Fractions containing the LGN/Gαi-1/NuMA complex were pooled (Complex Pool) and used for subsequent assays.