Briefly, larvae on day 1 of 3rd instar were individually fed with one piece of leaf fragment coated with different protein dosages of 2.5, 5 or 7.5 µg LMX fusion protein. After 72 h, ten larvae as a group were weighed and homogenized in 2 ml methanol. Ecdysteroids were extracted according to the method described by Hägele et al. [30] (link). The ecdysteroid measurement was carried out using High Performance Liquid Chromatography (HPLC) (More Biotechnology, China) based on the following program: Twenty microliters of the solution was injected into a LC-MS instrument (Waters e2695/2489, USA), separations were performed on a Waters Symmetry C18 column (4.6×250 mm i.d., 5 µm, Waters, USA) at a flow rate of 0.8 ml/min at 30°C, eluting with acetonitrile, methanol and water in a ratio of 1∶2∶4. α-ecdysteroid and β-ecdysteroid were used as standards. Thirty larvae were used in each treatment. All treatments were performed in triplicate. PBS buffer and 7.5 µg GST protein were used as controls.
E2695 2489
Manufactured by Waters Corporation
Sourced in United States
The E2695–2489 is a laboratory instrument produced by Waters Corporation. It is designed to perform high-performance liquid chromatography (HPLC) analyses. The core function of this product is to separate, identify, and quantify components within a liquid sample.
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Lab products found in correlation
5 protocols using e2695 2489
1
Ecdysteroid Quantification in Lepidopteran Larvae
2
Controlled Release Kinetics of KGN and SA
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The release experiments of KGN were carried out in 5 mL tubes. The scaffolds were placed in test tubes, 3 mL of PBS solution (pH = 7.4) was added and the tubes were placed in a constant temperature shaker at 37 °C. At each time point (1, 2, 3, 4, 6, 8, 10, 13, 16, 19 days), 1 mL of release solution was extracted from the tube and replaced with fresh PBS solution (n = 6). The concentration of the drug solution was determined by HPLC (High Performance Liquid Chromatography, e2695-2489, Waters, America). The formula for the cumulative release rate of KGN was as follows:![]()
where Wi represented the cumulative release of KGN at different times, and W0 represented the total load of KGN in the scaffold.
The release rate experiments of SA were like those of KGN. Briefly, the scaffold was placed in a centrifuge tube, and 3 mL of PBS solution (pH = 7.4) was added. The tubes were placed in a constant temperature shaker at 37 °C. At each time point, the 1 mL of release solution was collected from the tube and replaced with fresh PBS (n = 6). The concentration of the drug solution was determined by UVs (Ultraviolet-visible Spectrophotometer, UV2600, Shimadzu, Japan). The formula for the cumulative release rate of SA was as follows:![]()
where Wi represented the cumulative release of SA at different times, and W0 represented the total load of SA in the scaffold.
The release rate experiments of SA were like those of KGN. Briefly, the scaffold was placed in a centrifuge tube, and 3 mL of PBS solution (pH = 7.4) was added. The tubes were placed in a constant temperature shaker at 37 °C. At each time point, the 1 mL of release solution was collected from the tube and replaced with fresh PBS (n = 6). The concentration of the drug solution was determined by UVs (Ultraviolet-visible Spectrophotometer, UV2600, Shimadzu, Japan). The formula for the cumulative release rate of SA was as follows:
3
Chromatographic separation of compound 6
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Chromatographic separation of
compound6 and Imp-7–11 was achieved using a reversed-phase
column (C18, 4.6 × 250 mm, 5 μm, Waters e2695–2489
equipped with a 2414 RI detector) at a flow of 1 mL/min and a column
temperature of 40 °C. The mobile phase comprised acetonitrile,
water, and trifluoroacetic acid (70:30:0.1, v/v/v). The injection
volume was set as 50 μL.
compound
column (C18, 4.6 × 250 mm, 5 μm, Waters e2695–2489
equipped with a 2414 RI detector) at a flow of 1 mL/min and a column
temperature of 40 °C. The mobile phase comprised acetonitrile,
water, and trifluoroacetic acid (70:30:0.1, v/v/v). The injection
volume was set as 50 μL.
4
Identification of Vitamin C, Glucose, and Ethanol
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The identity of VC was confirmed by HPLC (Waters e2695–2489) with a Hypersil GOLDTM HILIC column (5 μm inner diameter, 250 μm by 4.6 μm, Thermo Scientific, catalog no. 26505–254630) with 85% acetonitrile, 14.985% H2O, and 0.015% phosphoric acid, a flow rate of 1 mL/min, and UV detection set at 245 nm, with pure VC as the standard (as shown in Supplementary Figure S3 ).
The identity of glucose and ethanol was confirmed by HPLC with an Aminex® HPX-87H Ion Exclusion column (9-μm inner diameter, 300 μm by 7.8 μm, Bio-Rad, catalog no. 125–0140) with 5 mM H2SO4, a flow rate of 0.6 mL/min, at 65°C with glucose⋅2H2O and absolute ethanol as the standard.
The identity of glucose and ethanol was confirmed by HPLC with an Aminex® HPX-87H Ion Exclusion column (9-μm inner diameter, 300 μm by 7.8 μm, Bio-Rad, catalog no. 125–0140) with 5 mM H2SO4, a flow rate of 0.6 mL/min, at 65°C with glucose⋅2H2O and absolute ethanol as the standard.
5
Chromatographic Separation of Compounds 4 and 5
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Chromatographic separation of
compounds4 and 5 and Imp-1–6 was
achieved using a reversed-phase column (C18, 4.6 × 250 mm, 3.5
μm, Waters e2695–2489 equipped with a 2414 RI detector)
at a flow of 0.8 mL/min and a column temperature of 40 °C. The
mobile phase comprised monopotassium phosphate solution (3.4 g/L),
acetonitrile, and phosphoric acid (30:70:0.1, v/v/v). The injection
volume was set as 30 μL.
compounds
achieved using a reversed-phase column (C18, 4.6 × 250 mm, 3.5
μm, Waters e2695–2489 equipped with a 2414 RI detector)
at a flow of 0.8 mL/min and a column temperature of 40 °C. The
mobile phase comprised monopotassium phosphate solution (3.4 g/L),
acetonitrile, and phosphoric acid (30:70:0.1, v/v/v). The injection
volume was set as 30 μL.
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