Agilent 1260 infinity 2 hplc

SEC-MALS was performed to assess the oligomeric state of rXKR9. Ten micrograms of of purified rXKR9 was filtered through a 0.1 μm filter, injected onto a Superdex 200 Increase 3.2/300 GL column, equilibrated in LMNG SEC buffer, and run at room temperature on an Agilent 1260 Infinity II HPLC coupled with an Eclipse3 system equipped with a miniDAWN TREOS MALS detector and Optilab T-rEX refractometer (Wyatt Technology). Data was analyzed in the ASTRA software package. The dn/dc values used were 0.19 ml/g for rXKR9 and 0.132 ml/g for LMNG.

One gram of pulverized sample was extracted twice with 30 ml of methanol in a KQ-300VDV ultrasonic cleaner (Kunshan Ultrasonic Instruments, Kunshan, China) at 45 kHz, 50°C, and 210 W for 30 min; and the extract was obtained by centrifugation at 12,000 g for 20 min. The total phenolic content was analyzed using the Folin–Ciocalteu method as described by Li et al. (2019) (link) using gallic acid as a standard, and phenolic contents were expressed as grams of gallic acid equivalents per kilogram of dry weight (g GAE kg^–1). For quantitative analysis of individual phenolic compounds, the extraction was filtered through a 0.45 μm filter, and the contents of individual phenolic compound were determined using an Agilent 1260 Infinity II HPLC equipped with Zorbax SB-C18 column (4.6 × 250 mm, 5 μm, Agilent) and UV/diode array detector (DAD) as described in our previous work (Dong et al., 2019 ). Calibration curves were generated using the standard solutions of individual phenolic compounds by plotting the peak area versus concentration, and the contents of gallic acid, kaempferol, protocatechuic acid, quercetin, rutin, and syringic acid were quantified by external standard method and based on dry weight, respectively.

The lycopene yields at different conditions were measured. Specifically, the bacteria cells cultured for 96 h at different conditions were harvested by centrifugation at 5000 × g for 5 min. The collected bacteria were washed twice with 1.0 g L^–1 NaCl. The cells were dried at 70 °C overnight to remove the water. Subsequently, lycopene in R. palustris bio-actuator was extracted with 3 mL mixed reagent of n-hexane and methanol (1:1 v/v). The mixed solution was vortexed for 5 min and then centrifugated at 7500 × g for 10 min at 4 °C. Finally, the exacted lycopene in supernatant was collected. The concentration of lycopene was measured by the Agilent 1260 Infinity II HPLC system (Agilent, Palo Alto, CA, USA). This involved a C18 column (4.6 × 100 mm, Agilent) with the mobile phase consisted of acetonitrile/methanol/chloroform (42.5:42.5:15 v/v/v). The flow rate was set at 1.0 mL min^–1 and the injection amount was set at 10 uL. The final concentrations of CCCP, rotenone, AQDS and HA used in this manuscript are 1 μM, 100 μM, 250 μM and 0.5 mg/mL respectively.

The organic acids in samples were analyzed by using Agilent 1260 Infinity II HPLC equipped with a DAD detector (Agilent Technologies Inc., Santa Clara, CA, USA). Separation and quantification of organic acids were performed at 210 nm with an Agilent Poroshell 120 EC-C18 column (4.6 × 150 mm, 4 μm). Two eluents, filtered through a 0.45 μm durapore membrane pore filter were used as mobile phases: eluent A: 0.5% (w/v) KH₂PO₄ (pH 2.3), eluent B: methanol, the elution condition was isostatic elution with A:B of 97:3 (v/v). Injection volume was 10 μL, flow rate was 0.7 mL/min, and column temperature was 35 °C.

iTRAQ-based proteomics analysis (Genechem Co.,Ltd., Shanghai, China) were performed to screen out the potential downstream target molecules of YKL-40. Three biological replicates were used for global proteome analysis. BMDM was treated with YKL-40 (500 ng/ml) or IgG as a control for 24 h to assay the potential pathways involved. We collected 20 mg protein from each sample and loaded into 12% SDS-PAGE for electrophoresis. The samples then underwent filter-aided sample preparation and were dissolved in 5X dissolution buffer. Then 100 μg peptide fragments from each sample were labeled with iTRAQ-8 plex reagents according to the protocol from AB SCIEX. The labeled peptides were separated by high-performance liquid chromatography (Agilent 1,260 Infinity II HPLC), followed by mass spectrometric analysis and identification. The raw data were processed with Mascot 2.5 software and Proteome Discoverer 2.1 to search the database. The experiment had been performed in our previous research, and the results were further explored in this study. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with identifier PXD028305 via the PRIDE partner repository.

The HPLC-separation of red wines conducted as described above afforded three fractions for each wine. The first fraction was collected from 15 to 25 min (fraction 1), the second one from 25 to 30 min (fraction 2) and the third fraction (fraction 3) from 30 min through the end of the chromatographic run (45 min). Each collected fraction was dried, solubilized in methanol and analyzed by HR-ESIMS in continuous flow injection in the positive ion mode. HR ESIMS experiments were performed on an Agilent 1260 Infinity II HPLC quaternary system coupled to a linear ion trap LTQ Orbitrap XL hybrid Fourier transform MS (FTMS) instrument equipped with an ESI ION MAX source (Thermo-Fisher). The following source settings were used (mass range m/z 100–2000): spray voltage 4.5 kV, capillary temperature 300 °C, capillary voltage 15 V, sheath gas 20 and auxiliary gas 21 (arbitrary units), tube lens voltage 140 V, and 25% collision energy. Calculation of elemental formulae was conducted by using Xcalibur software v 2.0.7 with a mass tolerance constrain of 5 ppm.

GXDK6 was incubated for 16 h under 0, 5, and 10% NaCl stress, respectively. The cells were collected by centrifugation at 8,000 × g for 10 min. The total proteins in GXDK6 were extracted by SDT lysis methods (Zhu et al., 2014 (link)). Proteome sequencing and analysis of the extracted proteins were conducted using a tandem mass tag (TMT)-based quantitative proteomics (Myers et al., 2018 (link)). TMT-labeled peptides were fractionated by RP chromatography using Agilent 1260 infinity II HPLC. LC–MS/MS analysis was performed on a Q Exactive plus mass spectrometer (Thermo Fisher Scientific) that was coupled to Easy nLC (Thermo Fisher Scientific) for 60/90 min. The MS/MS raw files were processed using MASCOT engine (Matrix Science, London, United Kingdom; version 2.6) embedded into Proteome Discoverer 2.2, and searched against the NCBI/NR/UniProt database. Proteins with fold change>1.2 and p value (Student’s t test) <0.05 were considered as differentially expressed proteins (DEPs).