Coomassie blue

Zymography was used to analyse gelatinase activity in DC crush sites from PBS-, Gel + PBS- and Gel + Decorin-treated animals, based on previously published methods^{36 (link),47 (link),81 (link)}. Briefly, the DC crush site and tissue to 3 mm either side of the lesion site from n = 4 rats/group (3 independent repeats, total n = 12 rats/group) were pooled together, homogenised without protease inhibitors and 40 μg of total protein was resolved on 10% minigels incorporated with gelatin substrates (Invitrogen, Paisley, UK) and clear protein lysis bands on a Coomassie Blue stained background were visualised according to the manufacturer’s instructions (Invitrogen). Bands were scanned using Adobe Photoshop and densitometry was performed in ImageJ (NIH Image) using the built-in gel plotting macros.

The gelatinolytic activities of MMP-2 and MMP-9 in the conditioning culture medium were assayed by electrophoresis on 10% polyacrylamide gels containing 1 mg/mL gelatin at 4°C. After electrophoresis, the gels were washed in 2.5% Triton X-100 for 1 h and incubated at 37°C for 24 h in activation buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10 mM CaCl₂, and 0.02% NaN₃). After staining with Coomassie Blue R-250 (10% glacial acetic acid, 30% methanol, and 1.5% Coomassie Brilliant Blue; Invitrogen Co., Carlsbad, CA, USA) for 2 h, the gels were destained with a solution of 10% glacial acetic acid and 30% methanol without Coomassie Blue for 1 h. White lysis zones, indicating gelatin degradation, were revealed by staining with Coomassie Brilliant Blue R-250 [41 (link)].

90 μg of trichloroacetic acid (TCA) precipitated M. ulcerans (NM20/02) protein lysate was resuspended in rehydration buffer (8 M urea, 2% 3-[(3-Cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 0.5% (v/v) ZOOM Carrier Ampholytes (Invitrogen), 0.002% bromophenol blue and 0.4% dithioerythritol (DTE)). The mix was incubated with a 3–10 pH gradient strip (ZOOM Strip; Invitrogen) over night (ON) at room temperature (RT). First-dimension isoelectric focusing (IEF) was performed on a ZOOM IPG runner (Invitrogen) using a step voltage protocol (175 V for 15 min, 175–2000 V for 45 min, 2000 V for 2 h). After IEF, the strips were incubated for 15 min with equilibration buffer (6 M urea, 50 mM Tris pH 8.8, 30% glycerol, 2% SDS, 30 mM DTE) followed by a 15 min incubation period with alkylating solution (6 M urea, 50 mM Tris (pH 8.8), 30% glycerol, 2% SDS, 0.23 M iodacetamide). Second-dimension gel electrophoresis was performed at 200 V for 35 min using a 10% NuPAGE Novex Bis-Tris ZOOM Gel (Invitrogen). The gel was stained with Coomassie blue (Invitrogen).

OMVs_Δ60 were prepared growing the E. coli BL21(DE3)Δ60 strain in an EZ control bioreactor (Applikon Biotechnology, Schiedam, The Netherlands) until the end of the exponential phase at 30 °C, pH 6.8 (±0.2), dO₂ > 30%, 280–500 rpm. OMVs were then purified and quantified as previously described [16 (link)]. Briefly, the culture supernatant was separated from biomass by centrifugation at 4000× g for 20 min. After filtration through a 0.22 μm pore size filter (Millipore, Burlington, MA, USA), OMVs were isolated, concentrated and diafiltrated from the supernatant using Tangential Flow Filtration (TFF) with a Cytiva Äkta Flux system (Marlborough, MA, USA). OMVs were quantified using DC protein assay (Bio-Rad, Hercules, CA, USA). OMV proteins were separated using a 4–12% gradient polyacrylamide gel (Invitrogen, Waltham, MA, USA) and finally stained with Coomassie Blue (Giotto, Sesto Fiorentino, Italy).

The purity of the rAAV preparations was determined following separation on SDS- PAGE gels and visualisation by Coomassie Blue (Invitrogen, CA) staining. The genomic titer of the vector preparations, the number of viral particles that contain a single-stranded AAV genome, was determined by standard quantitative real-time PCR42 (link). Briefly, the isolated rAAV particles were treated with DNAseI (15 min at 25 °C) prior to viral DNA extraction from 2 μl of the purified vector preparation. After the digestion of the viral capsid with proteinase K (60 min at 65 °C), the viral DNA was amplified with the rAAV specific primers WPREfor (ggc tgt tgg gca ctg aca at) and WPRErev (ccg aag gga cgt agc aga ag) using 2× Power SYBR master mix (Applied Biosystems, CA) on a real time PCR machine (Corbett RG 6000: 3 min at 95 °C followed by 40 cycles of 10 sec at 95 °C, 15 sec at 60 °C, 20 sec at 72 °C). A standard curve of diluted plasmid equivalent to 4.6 × 10⁹ to 1 × 10¹³ vg was used to determine virus titers of rAAV preparations. Titers for the various preparations that were used in the reported experiments are summarised in Supplementary table S1.

Plasmids for membrane scaffold protein (MSP) expression were a kind gift of Dr. Stephen Sligar (University of Illinois) through the Addgene program. MSP constructs were purified as described [63 (link)]. Briefly, the MSP-containing cell lysate was pelleted, and supernatant was filtered with a 0.45-μm filter before passage over a Ni-NTA column equilibrated with 1X PBS and 1% Triton X-100. The column was washed with lysis buffer, then with repeated washes of 40 mM Tris HCl, 300 mM NaCl, 1% Triton X-100, followed by washes with 1% 50 mM cholate (Sigma) and 20 mM imidazole pH 7.4, and finally 50 mM imidazole, with additional washes as necessary to clarify the resin. MSP was eluted in fractions of 40 mM Tris/HCl, 300 mM NaCl, 500 mM imidazole, pH 8.0, or an imidazole gradient as needed to achieve the desired purity of MSP. Fractions were checked by SDS-PAGE electrophoresis and Coomassie Blue staining (Invitrogen), and fractions containing MSP were collected and dialyzed overnight at 4°C in 20 mM Tris HCl, pH 7.4, 100 mM NaCl, 0.5 mM EDTA. Dialyzed MSP was collected and concentrated, then further purified by SEC as needed before plunge-freezing and storing at -80°C.

Expression of SUMO*-NS1 fusion protein was done in Spodoptera frugiperda (Sf)21 cells (Invitrogen). Sf21 cells were transfected using 3 μg of Bacmid DNA for high titer viral stock production, which was later used for infection of these cells at an MOI = 1. Since  pl-secSUMO* plasmid contains an upstream gp67 secretion signal of the SUMO* fusion which results in secretion of the SUMO*-rNS1 into the cell culture medium (Fig. 1), only culture supernatant of Sf21 cells was harvested on day 3 post infection. The culture supernatants (containing soluble SUMO*-NS1 fusion protein with 6xHis-tag) was concentrated (5X) and then purified by immobilized metal affinity chromatograph (IMAC) under native conditions. The purity as well as the molecular size of the fusion protein was determined by Coomassie blue stained 12% SDS-PAGE gel (Invitrogen) under both reducing and non-reducing conditions, and confirmed by western blot assay. The proper folding of the SUMO* tagged DENV-4 rNS1 protein was confirmed by commercial serotype cross-reactive Dengue Early (Panbio), which comprises MAbs reactive to the native hexameric NS1 in serum samples of dengue patients.Fig. 1

SUMO*-DENV-4 NS1 fusion gene construct. Schematic showing the DENV-4 fusion construct within the SUMO* cloning vector used to express recombinant protein in Spodoptera frugiperda (Sf)21 cells