Supplementary Figure 3: Comparison of the recovery efficacies of cross-linked APC/C complex by in-house-generated Ilama polyclonal antibodies, the LaG-16 and the LaG-16 lysine-less nanobody (2K/R) after on-bead crosslinking followed by denaturing elution.
From: A strategy for dissecting the architectures of native macromolecular assemblies
(a) Silver stain of the on-bead cross-linked APC/C complex that can be eluted by denaturing buffer. Equal amounts of in-house-produced Ilama polyclonal antibodies (Poly), wild-type LaG-16, and lysineless LaG-16-2K/R (2K/R) nanobodies were conjugated in parallel to epoxy magnetic beads. The antibody-conjugated magnetic beads were treated by DSS cross-linker to block residual reactive amines and were subsequently used to affinity isolate APC/C complex (using GFP-tagged Cdc16). The isolated complex was cross-linked on-bead (0.1 mM DSS). After on-bead cross-linking, the cross-linked complex that was non-covalently bound to the nanobody resins was eluted by hot LDS buffer (heated at 85°). The resulting protein complex was subjected to SDS-PAGE. The efficiently cross-linked APC/C complex corresponds to the high-molecular-weight region (>350 kDa) of the gel. A fraction of each sample was visualized by silver staining.
(b) Comparison of the recoveries of the cross-linked APC/C protein complex that could be eluted from GFP antibody-conjugated magnetic resins. Gel regions corresponding to the GFP-tagged protein Cdc16 (~70–160 kDa for non-cross-linked APC/C complex or >350 kDa for cross-linked APC/C complex) were in-gel digested by trypsin and analyzed by LC-MS. The experiments were independently repeated three times using freshly prepared antibody-conjugated beads (biological repeats), and for each biological repeat three analytical repeats were performed. The relative intensities of the GFP-tagged protein Cdc16 (which represents the relative amount of the complex) from three different anti-GFP reagents were quantified by MaxQuant (version 1.5) using label-free quantification (LFQ). Relative intensities of cross-linked APC/C are normalized to the sum of the input intensities of 2K/R.
