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. 2017 May 5;429(9):1364–1380. doi: 10.1016/j.jmb.2017.03.023

Fig. 5.

Fig. 5

SFK SH2 monobody interactome analysis (a) Schematic representation of a TAP-tagged monobody and the purification method including two copies of the B1 domain of staphylococcal protein G (2xProt.G), TEV protease recognition site, streptavidin-binding peptide (SBP), and Myc-tag N-terminal to the monobody. (b) Immunoblot analysis of TAP of Mb(Lck_3) monobody complexes from Jurkat cells. TE, total extract; SN1, supernatant IgG beads; TEV, eluate after TEV cleavage; SN2, supernatant streptavidin beads; E1, eluate from streptavidin beads; BB, boiled streptavidin beads to control the efficiency of elution. The bait protein and the main target of Mb(Lck_3) were identified by immunoblotting using an anti-Myc or anti-Lck antibody, respectively. (c) Mb(Src_2), Mb(Yes_1), and Mb(Yes_3) monobody complexes after TAP (10% of the E1 fractions) from K562 cells, separated by SDS-PAGE and visualized with silver staining. (d) Mb(Lck_1) and Mb(Lck_3) monobody complexes after TAP (10% of the E1 fractions) from K562 and Jurkat cells, separated by SDS-PAGE and visualized with silver staining.