Instantblue protein stain

To visualize the degradation of collagen by MMPs we used SDS-PAGE. Collagen Type I (0.5 mg/mL) (from human, Sigma-Aldrich) was incubated overnight with MMP8 and MMP9 (final concentration, 10 µg/mL), in absence of presence of SSL1 and SSL5 (final concentration 10 µg/mL). After overnight incubation, 2 × sample buffer was added and samples were run on a 10% SDS-PAGE gel for 1 h at 200 V. Afterwards, the gel was stained with Instant Blue protein stain (Expedeon, Cambridgeshire, United Kingdom).

Fifteen microlitre of samples containing purified TS-SNAP-A_2A were mixed with 5 μL NuPAGE^TM LDS sample buffer and resolved on a NuPage^TM 4–12% Bis-Tris 15 × 1.0 mm well gel using NuPage^TM MOPs SDS running buffer. Gels were run for 50 min at 200 V. Samples were not boiled prior to gel electrophoresis. 5 μL PageRuler^TM Prestained Protein Ladder was used as the ladder. Gels were scanned on an Amersham Typhoon imaging system (GE Healthcare Life Sciences, Pittsburgh, PA) using Fluorstage and Cy5 670BP30 filter sets with PMT set to auto and pixel size to 200 μm. After fluorescence was measured, gels were stained with InstantBlue® protein stain (Expedeon, Cambridge, UK) overnight. Gels were washed twice with dH2O for 5 min before visualising using a standard smart phone camera.

To remove small molecules or compounds that may contaminate the protein samples for MS analysis, a total of 0.5 mg of purified GSMT or rGSMT was resolved via 12.5% SDS-PAGE (1.5-mm thick) and stained with InstantBlue™ Protein Stain (Expedeon, C.B.S. Scientific Co., Inc., USA). The GSMT and rGSMT bands were excised and further diced into small pieces approximately 1.0 mm³ in size to increase the surface area of each particle. All gel pieces were completely destained with 50% acetonitrile (ACN) in 25 mM ammonium bicarbonate, pH 8.5. ACN was then removed, followed by re-equilibration in 25 mM ammonium bicarbonate. The samples were treated with 30 mM DTT at 37°C for 1 h to reduce the protein and then treated with 60 mM iodoacetamide (IAA) at room temperature in the dark for 1 h for alkylation. The gel particles were dehydrated with 100% ACN and then freeze-dried. TPCK trypsin (1:50 w/w) (Pierce, Rockford, USA) was added to recuperate the original size and incubated at 37°C for 16 h, and the reaction was terminated by addition of 5% TFA/50% ACN. The resulting peptides were recovered twice with extraction buffer following incubation for 10 min in a sonication bath (Branson 1510, Branson Ultrasonic Corporation, Danbury, CT). Extracts were dried in a vacuum centrifuge and stored at −20°C.

Protein samples (800 ng) were mixed with a sample buffer containing 0.1% β-mercaptoethanol and resolved in NuPAGE 4–12% Bis–Tris Protein Gels (Invitrogen) and electrophoresed in a XCell SureLock Mini-Cell Electrophoresis System (Thermo Fisher Scientific) using a constant voltage (120 V) at room temperature for 2 h. Protein bands were visualized using InstantBlue Protein Stain (Expedeon). The gels were then scanned with an EPSON Expression 11000XL scanner (Epson), and images were collected/exported using SilverFast 8.0 software (Epson).

The recombinant plasmids were transformed to the E. coli strain Rosetta (DE3) expression host. Protein expression was performed by culturing plasmid-containing bacteria in 0.5 l LB medium to the optical density of 0.6 at 600 nm and induced with 1 mM IPTG. The culture was then incubated for 16 h at 20°C with orbital shaking. Bacterial cells were collected by centrifugation at 8,000 x g for 30 min and resuspended in lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 10 mM imidazole, 0.1% Triton X-100, and 5% glycerol) before disruption by ultrasonication. The soluble cell lysate was obtained by centrifugation at 18,000 x g for 30 min, 4°C, mixed with Ni-NTA affinity resins (Expedeon), and loaded onto a 5-ml Poly-Prep Chromatography Column. The unbound proteins were washed using Tris–HCl buffers with 25 mM imidazole and histidine-tagged protein was eluted from the column using Tris–HCl buffers with 250 mM imidazole. Purified protein was dialyzed against a solution containing 10 mM sodium phosphate buffer pH 7.4 and 150 mM NaCl. The molecular weight and purity of the purified proteins were analyzed using 12% (w/v) discontinuous polyacrylamide gels in Tris-glycine SDS running buffers. Gels were visualized after InstantBlue protein stain (Expedeon).