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. Author manuscript; available in PMC: 2011 Oct 1.
Published in final edited form as: Mol Microbiol. 2010 Sep 2;78(2):490–505. doi: 10.1111/j.1365-2958.2010.07346.x

Figure 3. Asp3 forms complexes with Asp1, Asp2, Asp3 and SecA2.

Figure 3

(A) Lysates of E. coli BL21 (DE3) co-expressing His6Asp3 and Flag-tagged Asp1, Asp2, or SecA2 were applied to Ni2+ agarose, followed by washing of the columns and elution of the retained material. Lysates of cells co-expressing His6Asp3 and GST served as negative controls for nonspecific binding. The various fractions (flow, last wash, elution) were probed by Western blotting for proteins co-purifying with His6Asp3. Eluted His6Asp3 was detected using anti-His6 antibody (lower panel) and co-purifying proteins were detected with anti-Flag, anti-SecA2 or anti-GST antibodies (upper panel).

(B) E. coli BL21 (DE3) lysates expressing MalE, flagAsp1, flagAsp2 flagAsp3 or MalE.SecA2 were electrophoresed in SDS-PAGE (4–12%) and either stained with Coomassie blue (left panel), or transferred to membranes and probed with His6Asp3 (right panel). Binding of Asp3 to the immobilized proteins was detected with anti-His6 antibody.