Novagen bca protein assay kit

Xylanase activity was measured with EnzChek^® Ultra Xylanase Assay Kit (Life Technologies) according to manufacturer’s instructions. Briefly, cells were grown for 21 hours, reaching similar densities, harvested and lysed with CelLytic^™ B Plus Kit (Sigma-Aldrich). Cell lysate and supernatant from cultures were diluted and 50 μL of dilutions were mixed with 50 μL of xylanase substrate working solution in flat-bottom 96-well microtiter plate (Greiner Bio-One). They were incubated at room temperature for 40 min and the release of reaction products was measured with the ELx808^™ Microplate Reader (BioTek) with the excitation at 360 nm and emission at 460 nm. Total protein content was measured with Novagen^® BCA Protein Assay Kit (Merck) and xylanase activity was normalized to it.

The extracted biofilm matrix (25 μL) was transferred to 96-well plates (triplicate) and 200 μL of reagent mixture of Novagen^® BCA Protein Assay Kit (Merck KGaA, Darmstadt, Germany) were added to each well. The solution was homogenized by vortex and incubated for 30 min at 37 °C. Then, the absorbance at 562 nm was determined using PBS as control. The amount of protein was extrapolated from a standard curve performed with standard BSA concentrations. The amount of protein was then normalized by dry weight of biofilm (mg protein/g biofilm) [8 (link)].

Virus-encoded BiKEs were purified from supernatants of infected Vero cells (vBiKEs). 6 × 10⁶ cells in 15 cm culture dishes were inoculated with respective MV-BiKEs at MOI 0.03 in OptiPRO SFM serum-free medium (Thermo Fisher Scientific), cultured at 37 °C, 5% CO₂ and monitored for syncytia formation. Supernatants were collected, clarified (2500 × g, 5 min, 4 °C), and passed through a 0.22 µm pore size filter (Merck) before vBiKEs were purified by affinity exchange chromatography via the His₆ tag. Ni-NTA Spin Columns or Superflow Columns (Qiagen) were used for small and large scale purification, respectively, following the manufacturer’s instructions. In brief, cell-free supernatants were applied to equilibrated columns allowing BiKE binding to the Ni-NTA resin. Columns were rinsed with wash buffers containing 10 mM and 20 mM imidazole. Immobilized BiKE proteins were recovered with elution buffer containing 500 mM imidazole. Eluates were washed with DPBS, concentrated in Amicon Ultra-15 Centrifugal Filter Units with Ultracel-10 membranes (Merck), and stored at −80 °C. Protein concentration in purified vBiKE aliquots was quantified by BCA assay using the Novagen BCA Protein Assay Kit (Merck) according to the manufacturer’s instructions.