Amicon ultra 0.5 ml 100 k

Reconstructed MexAB–OprM (~4 mg) was precipitated by mixing buffer D supplemented with 20% (v/v) PEG-3350 at a volume ratio of 1:2 and then centrifuging at 20,400 g for 30 min at 4 °C. After removal of the supernatant, 250 µL of 50 mM HEPES-K (pH 7.5) containing 20 mg of Amphipol A8–35 (Anatrace) was added to the pellet, and the sample was incubated at 4 °C for 4 h with gentle rotation. Subsequently, ~125 mg of Bio-Beads SM2 (Bio-Rad) was added to the sample, which was rotated at 4 °C overnight. The beads were removed with a poly-prep column (Bio-Rad). The sample was subjected to a Superose6 Increase 10/300 column (GE Healthcare) with 50 mM HEPES-K (pH 7.5). Peak fractions were concentrated with an Amicon Ultra-0.5 mL 100 K (Merck Millipore).

The plasma was cleared by ultra-filtration using a Millex-GV syringe filter unit (0.22 µm, PVDF). The cleared plasma (0.3 mL) was loaded onto a Protein G Sepharose 4 Fast Flow column (gel volume of 2 mL) and eluted with 10 mmol/L phosphate-buffered saline (pH 7.4) at 0.2 mL/min. The eluate was collected in a total of 18 fractions, each containing 0.2 mL. Seven fractions with high absorbance at 280 nm were collected and concentrated by using an Amicon Ultra-0.5 mL 100K (Merck KGaA, Darmstadt, Germany) according to the manufacturer’s instruction, and analyzed by electrophoresis on a Bolt 4%–12% Bis-Tris Plus gel.

The average particle size (Z_av) and polydispersity index (PI) were determined by photon correlation spectroscopy, using a ZetaSizer Nano ZS (Malvern Instruments; Malvern, UK). The surface charge, measured as zeta-potential (ZP), was determined by electrophoretic mobility using the same instrument.
Encapsulation of TH was measured indirectly by quantification of unloaded amount. Samples were diluted 1:10 in Milli-Q water:ethanol (90:10) and centrifuged (Centrifuge 5415C, Geratebau Eppendorf GmbH, Engelsdorf, Germany) for 10 min at 14,000 rpm, using Millipore filter device (Amicon^® Ultra, 0.5 mL 100 K, Merck Millipore Ltd., Carrigtwohill Co. Cork IRL, Darmstadt, Germany). The filtered fractions were quantified by HPLC, and the EE was determined by the Equation (1):
where Ci is the initial concentration of the active and Cs is the concentration of the unloaded amount found in the filtered fraction.
HPLC quantitative analysis was performed by reverse-phase high-performance liquid chromatography (HPLC) by a modification of the method described previously [36 ]. Studies were carried out in Acquity Waters System with UV detector, using a Kromasil^® column (C18, 5 μm, 150 mm × 4.6 mm), (Teknokroma, Barcelona, Spain). The mobile phase consisted of acetonitrile:water under gradient conditions of 30:70/58:42/30:70 during 20 min. TH was determined at wavelength of 275 nm.