Labsonic

Stock suspensions of TiO₂ NPs at 5 mg/mL were freshly prepared for each experiment, using the dispersion procedure DP1 developed as part of the FP7 project NanoTEST. For 1 mL of stock suspension, 5 mg of TiO₂ NPs mixed with 1 mL of 10% fetal bovine serum (FBS, Gibco) in PBS (phosphate buffered saline) in a glass tube was sonicated using an ultrasonic probe sonicator (Labsonic, Sartorius, Gottingen Germany) at 100 W for 15 min on ice/water. This suspension was added to cell culture medium. Serial dilutions were made in cell culture medium to obtain the full range of NP suspensions, from 3 to 75 µg/cm², which were then immediately added to cells.

Harvested cells are resuspended in lysis buffer containing 50 mM potassium phosphate (pH 7), 250 mM sodium chloride (NaCl) and 1 mM phenylmethanesulfonyl fluoride (PMSF) and disrupted by sonication (Sartorius LABSONIC) at 30% amplitude and 0.7 cycle in ice-bath. Cell lysate is centrifuged at 14000 g for 10 minutes at 4 °C and the expressed protein is obtained in the supernatant.
Supernatant containing the expressed protein is purified using Nickel-Nitrilotriacetic acid beads (Qiagen), pre-previously equilibrated with lysis buffer. Beads are washed with four column volumes of buffer containing 50 mM potassium phosphate (pH 7), 250 mM NaCl and 1 mM PMSF and 30 mM imidazole to remove non-specific binding. The recombinant protein is eluted with buffer containing 50 mM potassium phosphate (pH 7), 250 mM NaCl and 1 mM PMSF and 250 mM imidazole.
Protein concentration ~6.2 μM is used in 12% Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to check the purity after affinity chromatography. Imidazole is removed from the sample by buffer exchange using a spin concentrator (10 KDa cut-off, Amicon) and lysis buffer. Concentration of pure protein is determined using Beer-Lambert law and absorbance 280 nm (BioSpectrometer, Eppendorf). Extinction coefficient (ε₂₈₀) from Protparam51 tool (Expasy server) obtained for the construct sequence is 18450 M⁻¹cm⁻¹.

Production culture samples were normalized to an OD₆₀₀ 2, centrifuged for 10 min (10,000× g at 4 °C) and free-media pellet was resuspended in 300 µL extraction buffer (50 mM MOPS, 150 mM NaCl, 5 mM DTT, 10% (v/v) glycerol, pH 7.5). Cells were disrupted through an ultrasonicator (Sartorius Labsonic, Goettingen, Germany) and soluble and insoluble protein fractions were separated after 30 min centrifugation (10,000× g at 4 °C). Protein fraction samples were mixed with 2X loading buffer, heated at 95 °C for 5 min and analyzed through a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE).