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. 2012 Nov 15;8(11):e1003031. doi: 10.1371/journal.ppat.1003031

Figure 3. Semaphorin-7A and MTRAP form an equimolar complex.

Figure 3

(A) Size exclusion chromatography of purified His-tagged Semaphorin-7A and MTRAP, individually and as a complex. Semaphorin-7A (with Cd4-tag; top panel) and MTRAP (without Cd4-tag; middle panel) were analysed by SEC separately and as a complex (bottom panel). (B) Multiangle light scattering of purified His-tagged Semaphorin-7A and MTRAP. Semaphorin-7A (with Cd4-tag; blue) and MTRAP (without Cd4-tag; green) were analysed by SEC immediately followed by MALS. Peaks correspond to SEC elution as a function of time with absorbance at 280 nm on the right y-axis, and the horizontal lines indicate molecular mass on the left y-axis. (C) SDS-PAGE analysis of the Semaphorin-7A∶MTRAP complex. SEC fractions of the Semaphorin-7a∶MTRAP complex (Figure 3 A) were analysed by SDS-PAGE and visualised by Coomassie staining. The middle fraction, corresponding to the highest point of the peak, was used for amino acid analysis. (D) Amino acid analysis of the MTRAP and Semaphorin-7A complex reveals a 1∶1 binding stoichiometry. The fraction of the highest point of the SEC peak of mixed purified MTRAP and Semaphorin-7A was analysed by amino acid analysis. The amino acid composition agrees best with 1∶1 binding. Bars indicate mean ± SEM n = 3.