Lc 20 prominence hplc system

Extraction of hormones from seeds or leaves of salt-stressed and control plants, which were previously biostimulated or not with triacontanol, was carried out according to Trupiano and colleagues^{57 (link)}. For HPLC analysis, an LC-20 Prominence HPLC system was used (Shimadzu, Kyoto, JP); it comprised an LC-AT quaternary gradient pump, an SPD–M20A photodiode array detector, and an autosampler SIL-20 AH. The injection volume was set at 20 µl, and the sample separation was performed with a Gemini–NX C18 column (250 × 4.5 mm, 5 µm particle size) (Phenomenex). Hormone elution was carried out with a gradient of solution B (99.9/0.1 v/v acetonitrile/TFA) in solution A (99.9/0.1 v/v water/TFA), running at a flow rate of 1.5 ml/min, at 45 °C. Solution B ramped from 15 to 30% in 5 min, from 30 to 50% in further 5 min, and increased to 80% in 2 min; finally, the gradient returned to the starting conditions in 3 min. The compounds of interest were identified based on their retention time and the corresponding UV spectra as well as by comparison with standard abscisic acid (ABA) (Duchefa Biochemie, Haarlem, NL) and gibberellins (GAs) (Merck). The latter ones were also used to build up calibration curves in the 5–200 µg/ml range, at wavelengths of 254 nm for ABA and 205 nm for GAs.

SEC-MALS was used to characterize
the recombinant expressed SF
mutants in solutions relating to their purity, native oligomers or
aggregates and molar masses. Analyses were performed on an LC20 prominence
HPLC system equipped with the refractive index detector RID-10A, the
photodiode array detector SPD-M20A (all from Shimadzu, Japan), and
a MALS Heleos Dawn8C plus QELS detector (Wyatt Technology, U.S.A.).
A Superdex 200 10/300 GL column (Cytiva, U.S.A.) was used and equilibrated
with PBS plus 200 mM NaCl (pH 7.4) as running buffer. Experiments
were performed at a flow rate of 0.75 mL min⁻¹ at
25 °C and analyzed using the ASTRA 6 software (Wyatt Technology,
U.S.A.). Proper performance of the MALS was verified by the determination
of the molar mass of a sample of bovine serum albumin. Prior to analysis,
samples were thawed, centrifuged (16000 g, 10 min), and filtered by
a 0.1 μm Ultrafree-MC filter (Merck Millipore, Germany). A total
amount of 25 μg of the respective SF variant was injected for
each measurement.

Size-exclusion chromatography combined with multi-angle light scattering (SEC-MALS) was used to determine the purity, the aggregation behavior and the molar mass of CD19-AD2. Analysis was performed on an LC20 prominence HPLC system equipped with the refractive index detector RID-10A, the photodiode array detector SPD-M20A (all from Shimadzu, Japan) and a MALS Heleos Dawn8C plus QELS detector (Wyatt Technology, United States). A Superdex 200 10/300 GL column (GE Healthcare, United States) was used and equilibrated with PBS containing 200 mM NaCl (pH 7.4) as running buffer. Experiments were performed at a flow rate of 0.75 mL min^–1 at 25°C and analyzed using the ASTRA 6 software (Wyatt Technology, United States). Determination of the molar mass of a sample of bovine serum albumin was performed to verify proper performance of MALS. Prior to analysis, samples were thawed, centrifuged (16000 g, 10 min) and filtered using 0.1 μm Ultrafree-MC filter (Merck Millipore, Germany). A total amount of 24 μg CD19-AD2 was injected for each measurement.