Fucoidan samples (5–6 mg) were hydrolyzed in 1 mL 2 M trifluoroacetic acid (TFA) at 100 °C for six hours (in closed vials in a thermostatted water bath). After hydrolysis, the TFA was evaporated and the residual TFA was removed by the addition of 2.5% ammonium hydroxide (NH4OH). The hydrolysates were used to determine the sulfate content by the barium chloride (BaCl2) gelatin method [55 (link)]. 0.5% gelatin solution was prepared in warm water (60–70 °C). 0.5 % BaCl2 was dissolved in gelatin solution and then allowed to stand for 2 h at 25 °C, then centrifuged at 10,000× g for 10 min. 10 μL of hydrolysate solution was added to 160 µL trichloroacetic acid (TCA) and 100 µL BaCl2-gelatin reagent. The mixture was allowed to stand for 30 min. A blank was prepared with 170 µl TCA and 100 µL BaCl2-gelatin reagent. The released BaSO4 suspension was measured at λ = 360 nm in a microplate reader (TECAN Infinite 200, Salzburg, Austria) while using UV-transparent 96-well microplates (Corning®, Tewksbury, MA, USA). Potassium sulfate was used as standard to generate a (linear) standard curve for the sulfate response at 360 nm.
Uv transparent 96 well microplates
Manufactured by Corning
Sourced in Austria, Switzerland
UV-transparent 96-well microplates are laboratory equipment designed for use in various applications that require UV light transmission. These microplates feature wells that allow the passage of UV light, enabling researchers to conduct experiments and analyses that involve UV-based detection or measurement techniques.
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Lab products found in correlation
Infinite 200, by Tecan (1 mentions)
Pyrimidine, by Merck Group (1 mentions)
Agilent 7890a gas chromatography, by Agilent Technologies (1 mentions)
Hydroxylamine hydrochloride, by Merck Group (1 mentions)
Spark 20m microplate reader, by Tecan (1 mentions)
M200 spectrophotometer, by Tecan (1 mentions)
6 protocols using uv transparent 96 well microplates
1
Sulfate Content Determination in Fucoidan
2
Glyoxalase I Activity Assay
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Go1 activity was evaluated according to a previous method (Li et al., 2018) with some modifications. Briefly, the assay was carried out in Corning UV Transparent 96-well microplates with a microplate spectrophotometer. The reaction mixture (100 μL/well) contained 50 mM sodium-phosphate buffer (pH 6.6), 2 mM methylglyoxal, and 2 mM GSH (preincubated for 30 minutes at room temperature). Erythrocyte lysates from samples (30 μg per well) were added to the buffer. Linear formation of S-(D)-lactoylglutathione was monitored at 240 nm for 20 minutes at room temperature. One unit of Glo1 activity was defined as the amount of enzyme that catalyzed the formation of 1 μmol of S-(D)-lactoylglutathione per minute.
3
Screening of Anion Exchange Resins
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Thirty-two anion exchange resins (Figure S1 ) were screened in duplicate at two pH conditions (pH 7.0 and pH 8.0) with a step gradient of NaCl elution conditions, in 50 mM NaCl increments ranging from 100 to 500 mM NaCl, followed by a 1 M NaCl strip. The resin screen was performed using the TECAN Evo system (TECAN group, Männedorf, Switzerland) in conjunction with robocolumns containing 0.1 mL of each resin (Repligen, Waltham, MA). Each column was loaded with concentrated, buffer exchanged harvest in 25 mM phosphate, 25 mM HEPES, 50 mM NaCl at either pH 7.0 or pH 8.0 to 222 mg/mL-r as measured by OD280 at a 2-min residence time. The loading density was set to 222 mg/mL-r to ensure enough product would be loaded for analysis, based on an expected product titer ~ 20 mg/L. The elution fractions were collected in UV-transparent 96-well microplates (Corning, NY) and transferred to the in-line plate reader. The total protein content of each fraction was measured by pathlength-corrected A280 and the S_dF_2P content was measured by binding to a monoclonal antibody targeting the N-terminal domain on the Octet binding platform.
4
Screening of Anion Exchange Resins
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Thirty-two anion exchange resins (Figure S1 ) were screened in duplicate at two pH conditions (pH 7.0 and pH 8.0) with a step gradient of NaCl elution conditions, in 50 mM NaCl increments ranging from 100 mM to 500 mM NaCl, followed by a 1 M NaCl strip. The resin screen was performed using the TECAN Evo system (TECAN group, Männedorf, Switzerland) in conjunction with robocolumns containing 0.1 mL of each resin (Repligen, Waltham, MA). Each column was loaded with concentrated, buffer exchanged harvest in 25 mM phosphate, 25 mM HEPES, 50 mM NaCl at either pH 7.0 or pH 8.0 to 222 mg/mL-r as measured by OD280 at a 2-minute residence time. The loading density was set to 222 mg/mL-r to ensure enough product would be loaded for analysis, based on an expected product titer ~20 mg/L. The elution fractions were collected in UV-transparent 96-well microplates (Corning, NY) and transferred to the in-line plate reader. The total protein content of each fraction was measured by pathlength-corrected A280 and the S_dF_2P content was measured by binding to a monoclonal antibody targeting the N-terminal domain on the Octet binding platform.
5
Quantification of Lignin Composition
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Total lignin was measured by acetyl bromide method with slight modifications from (Foster et al., 2010a ). Briefly, 1 mL of 25% acetyl bromide (Sigma‐Aldrich) was added to 4–5 mg of protein‐free CWRs and incubated at 50 °C for 3 h; then the samples were cooled down on ice for 15 min and 2.5 mL of glacial acetic acid was added to stop the reaction. A volume of 400 μL 1.5 M NaOH and 300 μL of 0.5 M hydroxyl amine hydrochloride (Sigma‐Aldrich) were added to 300 μL of the samples and vortexed. 200 μL of the solution was pipetted into the Corning® 96‐well UV‐Transparent Microplates (Corning, Kennebunk) and the absorbance was recorded at 280 nm with Spark 20M microplate reader (Tecan, Männedorf). A blank with the reagents only was included to correct background absorbance. The extinction coefficient of 17.75 g−1/cm for grasses was used for the calculation of lignin content (Foster et al., 2010a ).
To determine the lignin monomeric composition, thioacidolysis (Rolando et al., 1992 ) was performed with 10 mg protein‐free CWRs by follow the procedure described previously (Zhao et al., 2023 (link)). For derivatization, 50 μL of pyrimidine (Sigma‐Aldrich) and N‐methyl‐N‐trimethylsilyl trifluroacteamide (Sigma‐Aldrich) was added and incubated for 5 h. Derivatized products were quantified with Agilent 7890A gas chromatography as described (Zhao et al., 2021 (link), 2023 (link)).
To determine the lignin monomeric composition, thioacidolysis (Rolando et al., 1992 ) was performed with 10 mg protein‐free CWRs by follow the procedure described previously (Zhao et al., 2023 (link)). For derivatization, 50 μL of pyrimidine (Sigma‐Aldrich) and N‐methyl‐N‐trimethylsilyl trifluroacteamide (Sigma‐Aldrich) was added and incubated for 5 h. Derivatized products were quantified with Agilent 7890A gas chromatography as described (Zhao et al., 2021 (link), 2023 (link)).
6
Spectrophotometric Enzyme Activity Assay
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For each replication, 10 mixed-sex insects were weighed and homogenized in Sorensen’s buffer (0.05 M; pH 7.4) in a 1:10 ratio. Thereafter, the homogenate was centrifuged (10,000 RPM, 10 min, 4 °C). Blind tests were prepared using buffers instead of homogenates. All measurements were performed with the Tecan M200 spectrophotometer (Tecan Group Ltd., Männedorf, Switzerland) in the Corning® 96-well UV-Transparent microplates (Corning, Tewksbury, MA, USA). For each well, five measurements in different regions were taken, and the obtained results were averaged. In the samples the protein content was determined using the Bradford method, and then the enzyme activity was converted into Δ/min/mg of protein [17 (link)].
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