Instantblue stain

The supernatant was mixed with Co-Talon agarose (Clontech, Palo Alto, CA, USA) and gently rotated overnight at 4°C. The bound resin was washed with buffer A and CFTR eluted with 100 mM imidazole. Elution fractions were then analyzed using 7.5% SDS–PAGE and InstantBlue stain (Expedeon, Cambridge, MA, USA). Samples containing the DIBMA–TSch–lipoprotein particles after buffer exchange with Zeba desalting columns (Thermo Scientific, Waltham, MA, USA) were used directly for the single channel measurements and cryo-EM.

Two microgram of each antibody sample was measured and loaded into each lane of the gel. 12% or 8% resolving polyacrylamide gel and 3% stacking gel were used. Samples were loaded at reducing (addition of dithiothreitol (DTT)) and non-reducing conditions (no addition of reducing agent). Electrophoresis was conducted at initial constant 80V till the bands moved into the resolving gel. Then the voltage was increased to 120V. The gel run was stopped when the dye front reached the bottom of the gel. The gels were then stained with Instant BLUE stain (Expedeon, United Kingdom) and destained with distilled water overnight.

Six times non-reducing SDS loading buffer (2 μl) was added to 20 μl (0.2 mg/ml) PLY samples from the DLS assays described above and were run on a 1.5% SeaPlaque® agarose (Lonza) SDS agarose gel (SDS-AGE) tricine gel in tricine buffer (25 mM Tris–HCl, 250 mM glycine, 1% SDS, pH 8.3) at 110 mA for 1 hour. The gel was partially dehydrated and stained with Instant Blue stain (Expedeon) and destained in 7% acetic aid, 10% methanol.
PLY crystals (not grown in the presence of the gold compounds) scooped from a 4 μl volume crystallisation hanging drop were washed with 100 μl of 20 mM HEPES, 150 mM NaCl, pH 7.2 and collected by centrifugation. The washing was repeated three times. Residual wash solution was removed and crystals were solubilised in 20 μl wash buffer and 4 μl 6 × SDS, non-reducing, loading buffer (375 mM Tris-HCl pH 6.8, 6% SDS, 48% glycerol and 0.03% bromophenol blue). The protein were boiled for 2 minutes at 95 °C and loaded onto SDS-AGE gel with 10 μl SeeBlue® Plus2 Pre-stained Protein Standard (Life Technologies) and 1 μg PLY monomer as size references.

Clinical isolate UL32-GFP-Merlin-WT and GFP-Merlin delta US2-11 strains were kind gifts from Dr. R. Stanton (University of Cardiff, UK). Primary antibodies used for the following proteins were obtained from commercial sources: IE1/2 (mouse monoclonal, 11–003, Argene), GFP (mouse monoclonal, 7.1 and 13.1, Roche), pp65 (mouse monoclonal, 1-L-11, Santa Cruz), anti-ubiquitin (mouse monoclonal, clone Ubi-1, Merck). The antibody for HA (12CA5) is a mouse hybridoma supernatant. Secondary HRP-conjugated goat anti-mouse/rabbit antibodies were from Jackson ImmunoResearch Laboratories. Purified anti-human leukocyte antigen (HLA-A,B,C) antibody (clone W6/32), and isotype control antibody (clone MOPC-173) were from Biolegend, anti-IFN-γ antibody (clone AF-285-NA) was from R&D systems. Antibodies against IFN-α (Clone MMHA-6) and IFN-β (Clone MMHB-3) were from PBL Assay Science and isotype control antibody (MOPC-21) was from Biolegend. Alexa Fluor 488-conjugated goat anti-mouse was from Invitrogen. ZVAD-fmk was from ENZO Life Sciences. Instant Blue stain was from Expedeon and MG132 was obtained from Sigma. Immunoblotted proteins were detected using Enhanced Chemiluminescence (ECL) [44 (link)] or ECL prime western blotting detection reagents (GE Healthcare) and ChemiDoc XRS+ (Bio-Rad).

6×His-4×SUMO2-Strep was biotinylated as described above. 30 μg of biotinylated protein were mixed with 60 μl of streptavidin agarose resin (Thermo Fisher) and rotated (end-over-end) for 30 min at RT. The 4×SUMO2-bound beads were then centrifuged and washed. 30 μg of 6×His-XRCC4 diluted in 500 μl of PBS containing 1% Igepal were added to the 4×SUMO2-captured beads and rotated (end-over-end) for 1 h at RT. Subsequently, the beads were washed 5 times in PBS containing 1% Igepal, and resuspended in 2× SDS Laemmli buffer (120 mM Tris–HCl pH 6.8, 4% SDS, 20% glycerol, 0.02% bromophenol blue and 2.5% β-mercaptoethanol). Proteins were visualised with InstantBlue stain (Expedeon).

To analyze the amount of DDB complex formation by size exclusion chromatography (SEC), 500 nM fully purified dynein, 1 μM pig dynactin and 4 μM SNAPf-BICD2N (dimer) were mixed in a volume of 80 μL and incubated on ice for 15 min before SEC was carried out as described above. The elution volume of each species was determined by running fractions corresponding to different peaks on SDS-PAGE (Novex 4%–12% Bis-Tris precast gels run in MOPS buffer - Life Technologies) stained with Instant Blue Stain (Expedeon) or SYPRO-Ruby (BioRad), according to manufacturer’s instructions. The elution volume individual components (dynein, dynactin and BICD2N) was confirmed by separate SEC runs. Running the same gel in MES buffer allowed visualization of individual dynein light chains.
For preparation of DDB samples for electron microscopy, 270 nM dynein, 540 nM dynactin and 5 μM GFP-BICD2N were diluted to 100 μL in GF150 buffer and incubated on ice for 15 min. 10 μL of 0.1% (v/v) glutaraldehyde (Sigma-Aldrich), diluted in GF150 was added and incubated for a further 15 min. The cross-linking reaction was quenched by addition of 10 μL 1M Tris-HCl pH 7.4 before SEC. The DDB containing fractions (identified as above but using SYPRO Ruby Gel Stain (Bio-Rad)) were pooled and concentrated to a volume of 30 μl.