Hemolymph was collected after cutting the first abdominal prolegs of mutant L5 larvae. A few phenylthiourea (Sigma-Aldrich Korea, Seoul, South Korea) granules were added to the hemolymph sample to prevent coagulation. After centrifugation at 400 × g for 3 min, the supernatant plasma was diluted 20× with distilled water. The diluted plasma sample was cleaned with a Sep-Pak C18 cartridge (Walters Associates, Milford, MA, United States). Trehalose was identified and quantified using high-performance liquid chromatography (HPLC) (BioLC, Dionex, Sunnyvale, CA, United States) with the main column (CarboPacMA1, 4 × 250 mm, Dionex) and a guard column (CarboPacMA1, 4 × 50 mm, Dionex) following the method described by Park and Kim (2013) (link). The injection volume of the sample was 25 μL. NaOH (400 mM) was used as an elution buffer at a constant rate of.4 mL/min. Pulse amperometry mode of an electrochemical detector (ED40, Dionex) was used for detecting trehalose and other sugars.
Biolc
Manufactured by Thermo Fisher Scientific
Sourced in United States
The BioLC is a high-performance liquid chromatography (HPLC) system designed for the analysis and purification of biomolecules. It is capable of performing a variety of analytical and preparative techniques, including size-exclusion chromatography, ion-exchange chromatography, and affinity chromatography.
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Lab products found in correlation
5 protocols using biolc
1
Trehalose Quantification in Larval Hemolymph
2
Analysis of Cello-oligosaccharides using HPAEC-PAD
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Reaction products were analyzed by high-performance anion exchange chromatography (HPAEC) with pulsed amperometric detection (PAD) following (Isaksen et al. 2014 (link)) with minor modifications. The HPAEC system (BioLC, Dionex, Sunnyvale, CA, USA) was equipped with a electrochemical gold electrode and CarboPac PA1 analytical column (250 mm × 2 mm i.d., Dionex). Ten microlitre reaction samples were injected, and eluted using a gradient of 0.1 M NaOH (eluent A) and 1 M NaOAc in 0.1 M NaOH (eluent B). Solute elution was performed at 0.3 mL/min with initial conditions set to 0.1 M NaOH (100%). A stepwise linear gradient was applied as follows: a 10 min linear gradient from 100% eluent A (starting condition) to 10% eluent B, a 15 min linear gradient to 30% eluent B, and a 5 min gradient to 100% eluent B. The column was reconditioned between each run by running initial conditions for 10 min. Non-oxidized cello-oligosaccharides were used as standards.
3
High-Performance Anion-Exchange Chromatography
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The HPAEC system consists of a Dionex Bio-LC gradient pump with GM-3 (4 mm) gradient mixer, CarboPac PA-100 column (4 × 250 mm), and an electrochemical detector with AgCl as reference electrode. The waveform was carbohydrates (standard Quad), the following pulse potentials were used for detection: t = 0 s, E = 0.10 v; t = 0.20 s, E = 0.10 v; t = 0.40 s, E = 0.10 v; t = 0.41 s, E = − 2.00 v; t = 0.42 s, E = − 2.00 v; t = 0.43 s, E = 0.60 v; t = 0.44 s, E = − 0.10 v; t = 0.50 s, E = − 0.10 v. The sample injection volume is 20 µL and column oven temperature is maintained at 30 °C.
4
Quantitative Analysis of Cello- and Chito-Oligosaccharides
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For qualitative analysis, samples were analysed by MALDI-TOF MS, as previously described2 (link). For quantitative analysis, cello-oligosaccharides (native and oxidized) were separated by high performance anion exchange chromatography (HPAEC) and monitored by pulsed amperometric detection (PAD) using a Dionex Bio-LC equipped with a CarboPac PA1 column as previously described61 (link). Chromatograms were recorded and analyzed using Chromeleon 7.0 software. Oxidized dimers and trimers were quantified using GlcGlc1A and (Glc)2Glc1A standards obtained by incubating (40 °C, 1000 rpm) cellobiose (2 mM) or cellotriose (2 mM) with the cellobiose dehydrogenase from Myriococcum thermophilum (MtCDH,62 (link) 2 μM, 3 successive additions every 24 h to obtain maximum conversion of 95%). Chito-oligosaccharides resulting from the action of SmAA10A on β-chitin were analyzed using a Dionex Ultimate 3000 UHPLC system equipped with a Rezex RFQ-Fast acid H+ (8%) 7.8 × 100 mm column as previously described45 (link). The elution of chitooligosaccharides was monitored using a UV detector (194 nm). Prior to analysis of solubilized mixtures of chitooligosaccharides, these chitooligosaccharides were hydrolyzed with a chitobiase, SmGH20, from S. marcescens (1 µM final concentration) yielding chitobionic acid as the only oxidized product10 (link).
5
HPLC-based Glucose and Organic Acid Analysis
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The concentration of glucose was determined using a HPLC system (BioLC; Dionex, CA, USA) coupled to an electrochemical detector (ED40; Dionex, CA, USA) and CarboPac MA-1 column [25] . A 600 mM NaOH solution was used as the eluent. Organic acids were measured by high-pressure liquid chromatography over an organic acid column (Aminex HPX-87H; Bio-Rad) at 60 o C with 0.1 N sulfuric acid as the eluent and a flow rate of 0.6 ml/min. The peaks were detected by determining the UV A 210 .
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