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. 2015 Oct 30;5:15774. doi: 10.1038/srep15774

Figure 2. Defining the Ppz1/Hal3 complex stoichiometry.

Figure 2

(a) Increasing amounts of recombinant Hal3 were incubated with GST-Ppz1Cter bound to glutathione-agarose beads. After recovering the flow-through and washing, the beads were resuspended in Laemmli buffer, boiled, and aliquots (60 μL) of the supernatant analyzed by 8% SDS-PAGE followed by Coomassie staining. The upper panel shows the amounts of Hal3 bound by GST-Ppz1Cter, whereas the lower panel illustrates the amounts of unbound Hal3 present in the flow-through (40 μL samples). The first 2 lanes include samples (2 μg) of GST-Ppz1Cter and Hal3 proteins used in the experiment (note that the band present in the GST-Ppz1Cter sample that is similar in size to Hal3 is in fact the E. coli DnaK chaperone, as established by mass spectrometry analysis). Bands corresponding to Hal3 are identified with a closed arrow; GST- Ppz1Cter with an open arrow. Molecular weight (MW) is in kilodalton (kDa). (b) The relative ratio bound Hal3/GST-Ppz1Cter was obtained by scanning the stained gels and integrating the relevant signals with Un-Scan-It Gel (Silk Scientific) software. Data represents the mean ± SD from two independent experiments. (c) 6 × His-Ppz1Cter and Hal3 were co-expressed from plasmid pETDUET-1, the complex recovered by Ni2+-affinity chromatography and concentrated (“sample” in panel D). The sample was subjected to SEC (Superdex 200) and the shown A280 elution profile was recorded. Arrows indicated the void volume (V0) and the elution volumes (Ve) of known proteins: bovine serum albumin (BSA, 67 kDa), ovalbumin (Ovo, 43 kDa) and cytochrome C (Cyt.C, 12.3 kDa). The elution volumes of recombinant Ppz1Cter (42.3 kDa) and Hal3 (64 kDa) are also shown; note that the latter elutes with a Ve equivalent to that of a ~104 kDa protein. The numbers denote the fractions collected. (d) The indicated fractions (1 mL) in panel C were collected; 10 μL of each was subjected to 10% SDS-PAGE analysis and stained with Instant Blue (Expedeon). Note the similar intensity of the bands at ∼66 kDa (Hal3) (◀) and ∼40 kDa (6 × His-Ppz1Cter) (◁), indicating a 1:1 protein ratio in the eluted complex. MW, molecular weight markers.