Nupage lithium dodecyl sulphate sample buffer

Cultures were set up as described above and incubated for 5 days after which cells were collected by centrifuging 10 ml of culture at 4000 g for 10 min at 4°C. Cell pellets were placed on dry ice before storing at −20°C until further processing. The cell pellets were resuspended in 200 μl 1x NuPAGE lithium dodecyl sulphate (LDS) sample buffer (ThermoFischer Scientific), supplemented with 1% ß‐mercaptoethanol. Cell pellets were lysed by bead beating (2 x 45 s and 1 x 30 s at 6.0 m/s) and sonication (5 min), followed by three successive 5‐min incubations at 95°C with short vortex steps in between. Cell lysates containing all proteins were loaded on an SDS‐PAGE precast Tris‐Bis NuPAGE gel (Invitrogen), using MOPS solution (Invitrogen) as the running buffer. Protein migration in the SDS‐PAGE gel was performed for 5 min at 200 V, to allow removal of contaminants and purification of the polypeptides. The resulting gel was stained using SimplyBlue SafeStain (Invitrogen) to visualize the cellular proteome. The gel bands containing the cellular proteome were excised and stored at −20°C until further processing.

Peptide samples were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under denaturing and reducing conditions using 4–12% Invitrogen NuPAGE Novex Bis-Tris precast gels (Thermo Fisher Scientific, Loughborough, UK) and the Invitrogen XCell SureLock Mini-Cell (Thermo Fisher Scientific, Loughborough, UK). An aliquot of 10 μL peptide samples was incubated with 4 μL NuPAGE lithium dodecyl sulphate (LDS) sample buffer (Thermo Fisher Scientific) and 2 μL of 500 nM dithiothreitol (DTT) (Fluorochem, Hadfield, UK) for ten minutes at 95 °C and 15 μL of sample mixture loaded per lane and 5 μL of SeeBlue Plus2 pre-stained protein standard (Thermo Fisher Scientific, Loughborough, UK) loaded in at least one lane. The samples were run using 1X 2-morpholinoethanesulfonic acid (MES)-SDS running buffer (Thermo Fisher Scientific, Loughborough, UK). Electrophoresis was performed under a constant current of 125 mA for 40–60 min. The gel was stained using Biosafe Coomassie Brilliant Blue G-250 stain (Biorad, Watford, UK) for 60 min, washed and destained in water overnight and imaged using a Syngene G:box and GeneSnap software (Syngene, Cambridge, UK).

Clotting mixtures (20 μL) containing fibrinogen (0.25 mg/mL), zymogen FXIII (10μg/mL), thrombin (0.5 U/mL), and CaCl₂ (10 mM) were incubated at 37 °C for 0, 2, 5, 10, 15, 20, 30, 60, and 120 min. Reactions were stopped by adding 4× NuPAGE lithium dodecyl sulphate (LDS) Sample Buffer (ThermoFisher Scientific) and 10× NuPAGE Sample Reducing Agent (Thermo Fisher Scientific), immediately followed by heating at 90 °C for 10 min. Samples and molecular weight marker (Precision Plus Protein Dual Color Standards; BioRad) were run onto a NuPAGE 4 to 12% Bis-Tris Protein Gel (Thermo Fisher Scientific), and gels were stained using InstantBlue (Expedeon). Protein bands were visualized and quantified using Genesys and GeneTools softwares (Syngene). Band quantification for each lane was relative to the amount of B-β chain staining. Experiments were performed in triplicate.