Shim pack clc ods

The HPLC analysis was carried out with a modified C18 column 250 mm × 4.6 mm × 5 µm (Shim-pack CLCODS, Shimadzu, Canby, Oregon). The solvents used were (A) water acidified with 5% formic acid and (B) methanol HPLC grade. The elution gradient established was 15% B for 5 min, 15 to 80% B in 25 min, and maintaining 80% B isocratic for 15 min to rebalance the column, using a flow rate of 1.0 mL/min. The mass detection was performed in a positive mode with a capillary voltage of 2500 V; end plate offset: 2000 V; capillary output 110 V, skimmer 1 20 V, skimmer 2 10 V, dry gas (N₂) temperature 325 °C and flow 11 L/min, nebulizer 60 psi, sweep range from 200 to 800 m/z temperature set at 25 °C. Dual online DAD detection was performed using 280 and 520 nm as the wavelengths of choice.

5.0 × 10⁶ of EC2-1H9 cells or the engineered cells were seeded in T175 culture flask containing MEM-α supplemented with 10% (v/v) dFBS, 3.5 g/L glucose, 1% (v/v) Ab-Am solution, and 20 nM MTX. The culture medium was replaced with serum-free medium (CHO-S-SFM II; Gibco) in 3 days. After 2 days, the culture medium was collected, filtered using 0.45 μm filter (Sartorius, Göttingen, Germany), and dialyzed against phosphate buffer saline (PBS, pH 7.4) at 4 °C, overnight. To purify rhEPO, cultured medium was subjected to EPO Purification Gel (MAIIA Diagnostics, Uppsala, Sweden) according to the manufacturer’s instructions. Purified rhEPO was dialyzed against distilled water at 4 °C, overnight. The concentration was measured using a Quant-iT™ protein assay kit (Invitrogen) and stored at −80 °C until use.
The sialic acid level of the purified rhEPO was examined using the OPD-labeling method as previously described^{26 (link)}. Briefly, sialic acid moieties from the purified rhEPO were released using 0.5 M NaHSO₄ at 80 °C for 20 min. Released sialic acid was labeled with OPD (o-phenylenediamine-2HCl; Sigma) at 80 °C for 40 min. The level of OPD-labeled sialic acid was determined using C18 reversed-phase column (Shim-pack CLC-ODS; Shimadzu, Kyoto, Japan) with 474 scanning fluorescence detector (excitation at 230 nm and emission at 420 nm, Waters).

The green propolis extract employed was produced and kindly provided by the Apis Flora Company (Ribeirão Preto, Brazil; Patent no. PI 0405483-0, published in Revista de Propriedade Industrial n. 1778, Jan 2, 2005). The ethanol extract of propolis employed (EPP-AF) has a shelf life of four and a half years under stable conditions.
The propolis extract was analyzed in a high-performance liquid chromatography system (i-Series Plus; Shimadzu, Kyoto, Japan) equipped with a system controller, a quaternary pump, and a diode-array detector. The data were analyzed with LC solution software, version 1.21 SP1 (Shimadzu). As can be seen in Fig. 1, we used a 4.6 mm × 250 mm column with a particle diameter of 5 µm and a pore diameter of 100 Å (Shim-Pack CLC-ODS; Shimadzu).Figure 1

Fingerprint analysis of the ethanol extract of propolis. Chromatograms were plotted at 275 nm with reverse-phase high-performance liquid chromatography on a C18 column (4.6 mm × 250 mm; particle diameter, 5 µm; pore diameter, 100 Å) and gradient elution with methanol and acidic water (pH 2.7). The chromatographic profile includes the following compounds: (1) caffeic acid phenethyl ester (in approximately 15 min); (2) p-coumaric acid (in approximately 20 min); (3) trans-Cinnamic acid (in 35–36 min); (4) aromadendrin (in 38 min); and (5) artepillin C (in 61–62 min).

The statistical model and the optimization were experimentally validated by culturing the strain JAJ13 under unoptimized and optimized levels of variables at 28 ± 2°C for 10 days. After the incubation, the antibacterial substance was extracted with equal volume of ethyl acetate and the top organic layer was dried for further analysis. The dried ethyl acetate extracts were resuspended in methanol and assayed as above for antibacterial activity. In order to compare the secondary metabolite profile of strain JAJ13 on unoptimized and optimized medium, the resuspended fractions were also analyzed in HPLC (Shimadzu) over a shim-pack CLC ODS (4.6 × 15 mm) column with liquid pump-LC-6AD, system controller-SCL-6B, UV–Vis detector (195–700 nm)-SPD-6AV, data processor-CR-5A, mobile phase - Methanol: Milli Q water (65:35) flow rate⁻¹ ml min⁻¹ and wavelength–350 nm.

Chemotaxonomic analyses of strain AJ110941^P were performed according to the previously described methods^{33 (link)}. Briefly, the genomic DNA G + C content and major respiratory quinones were analyzed using LC10 HPLC system with a Shim-pack CLC-ODS (Shimadzu) and Zorbax SB-C18 (Agilent Technologies Palo Alto, CA, USA), respectively. Cellular fatty acid compositions were determined using a GCMS-QP2010 system (Shimadzu).