486 tunable absorbance detector

The analyses of the extracted homo PSt were performed on a system with an M510 pump, U6K universal injector, 486 tunable absorbance detector (all from Waters Corporation, Milford, MA, USA) and 250 dual refractometer/viscometer detector (Viscotek/Malvern Corporation). The separation was achieved over a set of three 5 µm Styragel columns (HR 2, 3 abnd 5, Waters Corporation) and calibrated with 17 narrow dispersity PSt standards with molecular masses between 0.162 kDa and 956 kDa.

The growth of P. tricornutum cells was determined by measuring the absorbance of the culture at 680 nm using a spectrophotometer (UV-5200, China). Cell numbers were also counted using a hemocytometer under a microscope.
For fucoxanthin quantification, algal cells were pelleted from an 80 mL culture by centrifugation at 12,000× g at 4 °C. Harvested cells were frozen-dried, weighed and then ground into fine powder. One mL anhydrous ethanol was added to each of the algal samples, and the extraction was performed by vigorous vortex for 3 times (30 s each). The extraction was repeated three times. After each extraction, the mixture was centrifuged at 13,000× g at 4 °C for 10 min and the supernatants were combined, filtered through a 0.22-µm pore size membrane (Sangon Biotech, Shanghai, China) and then stored at 4 °C in the dark for further analysis.
Carotenoids in the extract were separated by a 2695 high performance liquid chromatography (HPLC) Separations Module (Waters, Milford, MA, USA). Fucoxanthin was detected by monitoring the absorbance at 445 nm using a 486 Tunable Absorbance Detector (Waters). A YMC Carotenoid HPLC Column (250 mm × 4.6 mm with 5-µm particle size) (YMC, Kyoto, Japan) was used and the elution was performed using a gradient of methanol/water ratio from 90/10 to 100/0 over 30 min, followed by a further 20 min with 100% methanol.

To determine the HER2(scFv)-PE24B purity, the final product was analyzed by HPLC using a Protein-pak 300SW SEC 7.5 × 300 mm column. The column was equilibrated with at least 10 CVs of 1 × PBS buffer, pH 7.4, using a Waters 600 Controller connected to a Waters 486 Tunable Absorbance Detector and a Waters 717 Plus Autosampler from Waters Corporation (Milford, MA, USA). The protein was injected onto the column at a flow rate of 1 mL/min over 25 min. The protein elution peaks were detected at 280 nm and checked by SDS-PAGE. The HER2(scFv)-PE24B purity was also evaluated using a Silver Stain Plus kit. The reaction was completed by adding 5% acetic acid (v/v) for 15 min when the bands became visible.

AG (98%) was supplied by Sigma-Aldrich, USA. The other two phytochemicals (DDAG and NAG) were obtained from in-house standards collection. Solvents (AR grade) used for isolation and purification of the compounds were supplied by Fisher Scientific (UK). Silica gel (70–230 MESH) and 20×20 cm silica gel 60 F254-coated TLC plates were purchased from Merck (Darmstadt, Germany). In addition, HPLC grade solvents including methanol and acetonitrile were provided by Merck (Darmstadt, Germany). The HPLC system was supported by Waters™ and consisted of Waters™ 600 Controller pumps, Waters™ 717plus Autosampler injector with a capacity of 96 samples. LiChrocart® HPLC-Column RP-18 (150×4.6 mm, Merck, Germany) was used as the stationary phase. The isocratic mobile phase was implemented with acetonitrile- water (40∶60 v/v) and 0.1% (v/v) analytical grade phosphoric acid dissolved in ultra-pure water at a flow rate of 1 mL/min [28] . The water used in this research was purified using the MilliporeTM water purification system. Detection was done at 223 nm using Waters™ 486 Tunable Absorbance Detector (photodiode array detector).

The analyses of the extracted linear PSt were performed on a liquid chromatography line consisting of M510 pump, U6K universal injector, 486 tunable absorbance detector (Waters Corporation, Milford, MA, USA), and 250 dual refractometer/viscometer detector (Viscotek/Malvern Panalytical Ltd, Malvern, UK). The separation was carried out with a set of three 5 µm Styragel columns (HR 2, 3, and 5, Waters Corporation) and calibrated with 17 narrow dispersity PSt standards with molecular masses between 0.162 kDa and 956 kDa (Polymer Standards Service, Amherst, MA, USA).