Millex gv 0.22 μm filter

Sulfasalazine concentrations in plasma were measured using high-performance liquid chromatography (HPLC) as previously described with minor modifications4 (link). Briefly, a 100-μl plasma sample containing the internal standard (0.125 μg/ml of p-dimethylaminobenzaldehyde) was added to 300 μl of methanol. The mixture was stored on ice for 10 min, centrifuged at 10,000 × g for 2 min, and the supernatant was then passed through a microporous membrane filter (Millex-GV 0.22-μm filters, Millipore Corp., Bedford, MA, USA). Each 20-μl sample was subjected to a HPLC analysis. A variable wavelength ultraviolet detector was adjusted to 365 nm for sulfasalazine. The assay was carried out based on the peak area ratios of sulfasalazine to the internal standard. The calibration curves were linear over a concentration range of 0.1 to 80 μg/ml (r² > 0.999).

Erlotinib levels in homogenized tumor tissues were determined by reverse-phase high-performance liquid chromatography (HPLC) with UV detection at 345 nm. Separation was achieved on a Waters Symmetry C18 column (150 × 4.6 mm, 5.0 μm; Waters, Milford, MA) preceded by the use of a Symmetry C18 Guard column (3.9 × 20 mm). The mobile phase was 50 mM potassium phosphate buffer (pH 4.8) containing 0.2 % triethylamine and acetonitrile (60:40, v/v), with 1.0 mL/min flow rate at 25 °C. Sample pretreatment involved mixing 500 μL of tumor tissue homogenate with 80 μL of internal standard (70 μg/mL of midazolam in methanol) and 5 mL of tert-butyl methyl ether for 10 min. After centrifugation (650 g, 10 min, 4 °C), the organic top layer was transferred to a clean tube and dried under nitrogen gas at 37 °C. The residue was dissolved in 250 μL of mobile phase. The solution was centrifuged (4,000 g, 30 min) and the supernatant was passed through a microporous membrane filter (Millex-GV 0.22-μm filters, Millipore Corp., Bedford, MA). Insoluble materials were removed by filtration, and the filtrate was analyzed by high-performance liquid chromatography. The calibration curves were linear over a concentration range of 20–4,000 ng/mL (r2 > 0.998).

The D,D-carboxypeptidase activity for LMM-PBP4 of PAO1 was assayed in vitro by monitoring the appearance of the monomeric tetrapeptide (M4) in mixtures containing increasing concentrations of monomer disaccharide pentapeptide (M5) (14.6 μM to 227.0 μM) as substrate, purified enzyme PBP4HC (0.57 μM) and PBS 1X pH 8.0, in a final volume of 200 μl. Reaction mixtures were incubated at 37 °C for 150 min. D,D-endopeptidase activity was determined using increasing concentrations of the dimeric compound tetrapentapeptide (D45) (from 8.3 μM to 137.3 μM), PBP4HC protein (0.59 μM) and PBS 1X pH 8.0. Reaction mixtures were incubated at 37 °C for 110 min. All enzyme reactions were terminated by boiling the samples for 2 min; they were centrifuged at 14,500 rpm for 10 min, filtered (Millex-GV Filter 0.22 μm, Millipore) and analysed by HPLC. Apparent K_m and V_max values were obtained from double-reciprocal Lineweaver-Burk plots of the data. k_cat was determined as V_max/[E₀] (where [E₀] corresponds to the micromolar concentration of purified LMM-PBP4). Graphical and statistical analyses were performed using the GraphPad Prism® v5.01 programme (GraphPad Software, Inc.). The results correspond to the mean value of experiments done in triplicate.

Macromolecular peptidoglycan was re-suspended in 500 μl of 50 mM phosphate buffer pH 4.9 and digested with Cellosyl (Hoechst AG) 100 μg/ml final concentration at 37 °C overnight. The enzyme reaction was stopped by boiling the sample for 15 min in a water bath. Coagulated protein and insoluble contamination were eliminated by centrifuging in a MiniSpin® Plus (Eppendorf) at 14,500 rpm for 15 min at room temperature. Muropeptides contained in the soluble fraction were mixed with 1/3 volume of 0.5 M sodium borate buffer (pH 9.0) and reduced with excess sodium borohydride (NaBH₄) for 30 min at room temperature. The excess borohydride was neutralised with phosphoric acid (dilution 1:10) to pH 3–4. Finally the samples were filtered through Millex®-GV Filter 0.22 μm (Millipore) units and stored at −20 °C. Reduced muropeptides were separated and analysed by HPLC (Breeze™ 2 System, Waters). Elution products were detected at wavelength 204 nm and identified by the retention time obtained. The relative quantity of muropeptides present in each sample was determined by integration of their respective absorption areas (Breeze™ 2, Waters) and expressed as a molar fraction (mol%) of the total content. When required, the individual peaks were collected, vacuum dried and stored at −20 °C.