Quick coomassie stain

For identification of exocyst subunit interactions, SF9 cells at 1x10⁶ SF9 cells/ml were grown in 24 well round bottom plates (Qiagen) and infected with baculovirus of the indicated single subunits. After 2 days of infection, the cells were pelleted at 500xg for 10 min, the supernatant discarded and the pellet frozen in liquid nitrogen. Upon use, the pellet was thawed and resuspended in 2 ml of standard buffer (20mM HEPES, 250mM NaCl, 0.5 mM TCEP), sonicated, and clarified at 4 °C. 25 μl of glutathione agarose beads (MRC-PPU Reagents and Services) were added and incubated for 1-2 h at 4 °C. Beads were isolated by centrifugation and washed three times in standard buffer using MobiSpin Column F (MoBiTec GmbH). The washed beads were resuspended in 50 μl 1x SDS loading buffer, boiled at 95 °C, and separated by SDS-PAGE. Gels were stained with Quick Coomassie Stain (Generon) and scanned.

Samples were loaded, as stated, on 10% tris-glycine gels and run at 180 V and 40 mA for 100 min. The gels were then stained with Quick Coomassie Stain (Generon, Slough, UK) at room temperature overnight. Excess stain was removed through deionized water washes. Gels were viewed and captured by Criterion Stain Free Imager (Biorad, Watford, UK).

GST-tagged CIT-K fragments were incubated with 190 ng of recombinant human Aurora B (Invitrogen), 0.1 mM ATP (Sigma-Aldrich), 5 µCi of [γ-³²P] ATP (6000 Ci mmol⁻¹, 10 mCi ml⁻¹) (PerkinElmer) and kinase buffer (20 mM HEPES pH 7.5, 2 mM MgCl₂, 1 mM DTT) in a final reaction volume of 15 µl. After 30 min incubation at 30°C with constant agitation, 15 µl of 2× Laemmli sample buffer was added to stop the reaction. Samples were boiled for 10 min and loaded on a 4–20% Tris–Glycine precast gel (Thermo Scientific). Gels were stained with Quick Coomassie Stain (Generon) to check the protein loading and then proteins were transferred onto a nitrocellulose membrane using the iBlot Dry Blotting System (Invitrogen). Membranes were exposed to Kodak BioMax XAR Films (Sigma-Aldrich) at −80°C. The radioactive CIT-K in vitro kinase assay was performed as described above except that GST-tagged CIT-K and KD-CIT-K were incubated with GST-tagged Aurora B, Borealin and INCENP in a final volume of 25 µl for 1 h at 30°C with constant agitation, where 25 µl of 2× Laemmli sample buffer was added to stop the reaction. The non-radioactive CIT-K in vitro kinase assay was performed as above except using GST-tagged INCENP 783–918 as the substrate, with ATP at a final concentration of 0.5 mM.

Samples were mixed with 4xLDS sample buffer (Thermo Fisher), loaded onto a 3–8% Tris-acetate gel (Thermo Fisher) and separated at 150 V for 50 min. Gels were then either stained with Quick Coomassie stain (Generon) or transferred onto a 0.2 µm nitrocellulose membrane using a Trans-blot Turbo transfer pack (Bio-Rad). The antibodies used for western blotting were anti-His (Sigma), anti-Strep HRP conjugated (iba) and anti-Smc3 (Bethyl Laboratories). Primary antibodies were probed with anti-mouse HRP conjugated antibodies (Thermo Fisher).

Pfs25::SpyTag and SpyCatcher::HBsAg were conjugated as previously described (20 (link), 23 (link)). Briefly, SpyCatcher::HBsAg VLP were incubated with Pfs25::SpyTag, at different molar ratio (1.5, 0.5, or 0.1 to obtain a conjugation efficiency of >90%, ~50%, or ~10%, respectively), 3 h to over-night (O/N) at room temperature (RT), with 10× reaction buffer (40 mM Na₂HPO₄, 200 mM sodium citrate, pH 6.2). The reaction was then dialyzed with a 100 kDa cut-off membrane (Spectra-Por® Float-A-Lyzer® G2) four times against 1,000-fold excess TBS 1×, to remove unreacted Pfs25::SpyTag. After filter-sterilization (Corning® Costar® Spin-X® centrifuge tube filters, pore size 0.22 μm), the total concentration was determined by BCA (Pierce™ BCA Protein Assay Kit, ThermoFisher Scientific), and the preparations were stored at −80°C until use. The conjugation efficiency (% of total SpyCatcher::HBsAg coupled to Pfs25::SpyTag) was analyzed by densitometry (with ImageJ software) on SDS-PAGE (NuPAGE 4–12% bis-tris gel, ThermoFisher), after Coomassie staining (Quick Coomassie Stain, Generon).