Vivaspin 500 column

For the aspiration of broncheoalveolar lavage (BAL), trachea was exposed through a midline incision. Lungs were lavaged 4–5 times with 1 ml PBS injected through the trachea. BAL fluid was spun at 2000 rpm for 10 minutes. Supernatants were concentrated using Vivaspin 500 columns (5 kDa cutoff, VS0012, Sartorius) and were analyzed for various cytokines using ProcartaPlex® Multiplex Immunoassay from eBioscience by the Immunomonitoring Platform, SIgN, A*STAR, Singapore.

The biosynthesis and purification of CK2α-pAzF was performed as described before [42 (link)]. For the recombinant expression of CK2β^1-193-pAzF, plasmids pCK2β^{1-193,Y176Stop} and pEVOL-pAzF were used to transform E. coli BL21(DE3). CK2β^1-193-pAzF was obtained and purified referring to the process of CK2α-pAzF [42 (link)] with the exception that before purification the cell lysate was stirred overnight at 4 °C in order to extract CK2β^1-193-pAzF [54 (link)].
Purified CK2α-pAzF (130 µg/mL) in buffer P50 (25 mM Tris/HCl (pH 8.5), 50 mM NaCl) was incubated with 50 µM DBCO-Sulfo-Cy5 (Jena Bioscience, Jena, Germany) for 1 h in the dark at room temperature (RT). By SPAAC reaction the specific labeled CK2α-DBCO-Sulfo-Cy5 was obtained. For the site-specific labeling of CK2β^1-193, purified CK2β^1-193-pAzF in buffer P100 (25 mM Tris/HCl (pH 8.5), 100 mM NaCl) was treated with fluorescein alkyne (0.25 mM), TCEP (Tris(2-carboxyethyl)phosphine, 1 mM), TBTA (Tris(benzyltriazolylmethyl)amine, 0.17 mM) and CuSO₄ (1 mM) for for 1 h in the dark at RT. By CuAAC reaction the specific labeled CK2β^1-193-Flu was obtained. For MST measurements an additional ultrafiltration step using vivaspin500 columns (Sartorius, Göttingen, Germany) was used to remove unbound fluorophore and additives of the click reaction.

Uropathogenic Escherichia coli (UPEC) strain 536 (O6:K15:H31) [13 (link)] was grown to exponential phase (Optical Density at 600nm, OD600≈1.5) in 10mL of RPMI 1640 medium (Thermo Fisher Scientific) supplemented with 10μM FeCl₃ (RF), at 37°C with shaking at 200 r.p.m. and then diluted 1:100 in RF (OD600≈0.015) to be grown to stationary phase for ~16 hours overnight. After overnight incubation (OD600≈2.5) the culture was assessed for viability using the LIVE/DEAD BacLight kit (Thermo Fisher Scientific) using the manufacturers recommended protocol. OD readings were taken and cuvettes with a 1 cm path length.
Bacterial cells were removed by centrifuging twice at 7,000 xg for 10 min at 4°C, after which any residual cells were removed from the supernatant by filtration using 0.22μm PES filter (Merck Millipore). Supernatants were concentrated using 100 kDa Vivaflow 200 cassettes (Sartorius AG), which removes proteins/molecules under 100 kDa, and the vesicles pelleted by centrifugation at 75,000 xg for 2.5hr at 4°C. Vesicles were resuspended in 20mM HEPES or PBS, filter sterilised using a 0.22μm PES filter syringe and again concentrated using 100 kDa Vivaspin 500 columns (Sartorius AG) and stored at -80°C.

The purified protein kinase CK2α-pAzF (130 µg/mL) in buffer P50 was treated with DBCO545 (Jena Bioscience, Jena, Germany) or DBCO-Sulfo-Cy5 (Jena Bioscience, Jena, Germany) in a final concentration of 50 µM. After 1 h in the dark at room temperature (RT) the reaction solution with the obtained CK2α-DBCO-fluorophore was directly applied for SDS-PAGE, CE-measurements or flow cytometry. In case of MST measurements an additional ultrafiltration step using vivaspin500 columns (Sartorius, Göttingen, Germany) was established in order to remove the unbound fluorophore.
For the SPAAC reaction of CK2β-AT-pAzF on the surface of E. coli, cell density was set to OD₅₇₈ = 1 and the click reaction was performed with DBCO545 (50 µM) for 1 h in the dark at RT. Cells were washed three times with PBS to remove unbound DBCO545 and subsequently used for flow cytometry measurement.