The 2-AB-labeled glycan mixture was analyzed using a Shimadzu LCMS-IT-TOF MS system equipped with an RF-20A XS fluorescence detector (Shimadzu Corp.). Separation of 2-AB glycans by normal-phase HPLC was performed on an ACQUITY UPLC® BEH Glycan 1.7 μm column (2.1 mm I.D. × 150 mm L; Waters, MA, USA) with fluorescent detection at excitation and emission wavelengths of 330 and 420 nm, respectively. Samples (1 μL) were injected onto the column, which had been equilibrated with 100% solvent B (9.9% water in acetonitrile containing 0.1% formic acid). The 2-AB glycans were separated for 50 min at 40°C on a linear gradient of 0–100% solvent A (40% acetonitrile in water containing 0.1% formic acid) at a flow rate of 0.2 mL/min. The detailed settings of the MS device were as follows: detector voltage, 2.1 kV; CDL, 200°C; heat block, 200°C; Nebulizer gas, 1.5 L/min; negative ion mode auto scan, m/z 250–2000; MS2 CID energy, 50%. According to a previous report [34 (link)] and test runs, peaks observed at later retention time points were more likely to contain terminally sialylated complex glycans; thus peaks observed at later time points were selected to estimate glycan composition.
Rf 20a xs fluorescence detector
Manufactured by Shimadzu
Sourced in Japan
The RF-20A XS fluorescence detector is a versatile lab equipment designed for sensitive and precise fluorescence detection. It features high sensitivity, a wide dynamic range, and advanced optical systems to enable accurate and reproducible measurements. The core function of this detector is to quantify the fluorescence emission of sample analytes.
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Lab products found in correlation
Nexera x2 hplc system, by Shimadzu (2 mentions)
0.2 μm syringe filter, by Thermo Fisher Scientific (1 mentions)
Sil 20acxr autosampler, by Shimadzu (1 mentions)
Lc 20ad xr binary pump, by Shimadzu (1 mentions)
Cto 20ac column oven, by Shimadzu (1 mentions)
Spd m20a diode array detector, by Shimadzu (1 mentions)
Superose 6 increase 10 300 gl, by GE Healthcare (1 mentions)
High performance liquid chromatography system, by Shimadzu (1 mentions)
Lc 10a hplc system, by Shimadzu (1 mentions)
Tskgel amide 80 column, by Tosoh (1 mentions)
C30 column, by Shimadzu (1 mentions)
Qtof 6550, by Agilent Technologies (1 mentions)
Superdex 200 increase 10 300 sec column, by GE Healthcare (1 mentions)
Superdex 200 increase 10 300, by GE Healthcare (1 mentions)
Mediterranean sea ods2 column, by Teknokroma (1 mentions)
Prominence hplc system, by Shimadzu (1 mentions)
15 protocols using rf 20a xs fluorescence detector
1
Liquid Chromatography-Mass Spectrometry Analysis of 2-AB Labeled Glycans
2
Quantifying Tocopherol Homologs by HPLC
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Fat samples (50–60 mg) were dissolved in 10 mL hexane and filtered with a 0.2 μm syringe filter (Fisher Scientific, Pittsburgh, PA, USA). Each sample (20 μL) was then injected into a Shimadzu HPLC system equipped with a Supelcosil LC-Diol column (250 mm × 4.0 mm, 5 μm). The mobile phase was a mixture of hexane and dioxane (19:1 v/v), and the flow rate was 1 mL/min. A Shimadzu RF-20Axs fluorescence detector was used to detect tocopherol homologs at an excitation wavelength of 290 nm and an emission wavelength of 330 nm. Peak integration was performed using Shimadzu EZstart software (version 7.2). Tocopherol homologs in the samples were identified and their concentration calculated based on standard curves prepared with the individual tocopherol homologs.
3
Size Exclusion Chromatography of Fluorescent Proteins
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Example 5
Purified and dialyzed fluorescent protein samples were diluted into 50 mM Tris-HCl pH 7.5, 100 mM NaCl and filtered through 0.2 μm filters immediately prior to injection into a Shimadzu Nexera UHPLC equipped with a Waters BEH200 1.7 μm 4.6×150 mm size exclusion column and 4.6×30 mm guard column. Samples were run in the same buffer at a flow rate of 0.3 ml per minute for a total run time of 20 minutes. Fluorescence of the eluted protein was detected with an RF-20Axs fluorescence detector (Shimadzu) with 480 nm and 540 nm excitation and 530 nm and 620 nm emission wavelengths. Each LanYFP or variant sample was co-injected with mCherry [24], which had been purified under identical conditions and which served as a monomeric size standard. A control run of mCherry alone displayed no bleedthrough into the yellow emission channel.
4
High-Performance Liquid Chromatography Analysis
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HPLC was performed using a system equipped with a DGU-20A 3R degas unit, LC-20AD XR binary pump, SIL-20AC XR auto sampler, RF-20A XS fluorescence detector, SPD-M20A diode array detector, CTO-20AC column oven, and CBM-20A communications bus module connected to an LC work station (Shimadzu Corporation, Kyoto, Japan). A Cadenza CL-C18 column (ϕ 250 mm × 4.6 mm, 3 μm, Imtakt, Kyoto, Japan) was selected and a guard column (Cadenza CL-C18, ϕ 5 mm × 2 mm, 3 μm, Imtakt) was used to protect the analytical column.
5
Quantifying BEST1-Fab Binding Affinities
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To assess binding of the Fab to BEST1WT and BEST1S358E, 10 nM Fab was incubated (>30 min at 4°C) with various concentrations of BEST1 ranging from 1 to 200 nM in buffer (20 mM Tris-HCl, pH 7.5, 75 mM NaCl, 75 mM KCl, 1 mM DDM) containing either 5 mM EGTA or 10 µM CaCl2. 400 µl of each mixture was loaded onto an SEC column (Superose 6 increase 10/300 GL; GE Healthcare), which was equilibrated in the same buffer, and the fraction of unbound Fab was quantified from the area under the elution peak corresponding to free Fab (at 27.5 ml using tryptophan fluorescence on a Shimadzu RF-20AXS fluorescence detector), which is well separated from the peaks for BEST1 and BEST1-Fab complex (22.8 and 20.7 ml, respectively), in comparison to a Fab control. For high-affinity BEST1–Fab interactions (e.g., Kd < 50 nM), the binding of Fab to BEST1 will deplete the amount of free BEST1 in the assay at low concentrations of BEST1 (e.g., <50 nM), and thus, the actual binding may be tighter than indicated by a fit of the data (as described in Figs. 5 D and 6 D ). We indicate this by reporting Kd values as ≤X nM, where X is the value derived from the fit.
6
HPLC-DAD-Fluorescence Analysis
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A Shimadzu high-performance liquid chromatography (HPLC) system, combined with a Diode-Array Detection (DAD) detector and an RF-20A XS fluorescence detector (Shimadzu, Kyoto, Japan) were used.
7
HPLC Separation of 2-AB Labeled Glycans
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The 2-AB-labeled glycan mixture was analyzed using a Shimadzu LC-10A HPLC system equipped with an RF-20A XS fluorescence detector (Shimadzu Corp., Kyoto, Japan). Separations of 2-AB glycans by normal-phase HPLC were performed on a TSK gel Amide-80 column (5 μm, 4.6 × 250 mm; Tosoh Corp., Tokyo, Japan) with fluorescent detection at wavelengths of: Ex 330 nm and Em 420 nm. The samples were injected onto the column equilibrated with 36% solvent B (0.05% TFA in water) in solvent A (0.05% TFA in acetonitrile). The 2-AB glycans were separated with a linear gradient of 36–48% solvent B at a flow rate of 0.8 mL/min for 40 min at 45°C.
8
HPLC Analysis with Shimadzu Device
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High-performance liquid chromatography (HPLC) was performed with a Shimadzu device combined with a diode array detector and RF-20A XS fluorescence detector (Shimadzu, Kyoto, Japan).
9
Quantification of Tocopherols and Tocotrienols
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For quantification of tocopherols and tocotrienols, the oil was saponified, then the compounds were determined by the HPLC method as described by Ben Mohamed et al. [21 (link)] with minor changes. The contents of tocopherols and tocotrienols were measured by an LC-20A high-performance liquid chromatography (Shimadzu, Kyoto, Japan) using a C30 column (4.6 mm × 250 mm, 5 µm, Shimadzu, Kyoto, Japan) and an RF-20A xs fluorescence detector (Shimadzu, Kyoto, Japan).
10
Procainamide Labeling and HPLC Analysis of N-Glycans
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Purified N-glycans were labelled with procainamide (Supelco) according to the manufacturer’s instructions. Labelled N-glycans were resuspended in 10 µL solvent A (80 mM formic acid buffered to pH 4.4 with ammonia), and the injection volume was 1.5 µL. Separation of glycans was performed with an Agilent AdvanceBio Glycan Mapping column (2.1 × 150 mm, 1.8 μm) with a Security Guard Ultra precolumn (Phenomenex) on a Nexera X2 HPLC system with a RF-20Axs fluorescence detector equipped with a semimicro flow cell (Shimadzu, Korneuburg, Austria). Fluorescence was measured with wavelengths Ex/Em 310 nm and 370 nm. The applied gradient started with an initial hold of solvent B (85% acetonitrile in solvent A) at 99% for 8 min and a decrease to 57% solvent B over 60 min, followed by 25% solvent B in 2 min at a flow rate of 0.4 ml/min. The PGC analysis of purified N-glycan isomers was done as described previously [24 (link)].
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