Transglucosylation products were analyzed using HPLC under the following conditions. For the degree of polymerization (DP) analysis, the HPLC system was used with an MCI GEL CK04S column (Mitsubishi Chemical, Japan) at 65 °C and a refractive index detector. To determine the DP of each peak, analysis was carried out in the same manner as in the DP analysis except that ELSD and LC MS were connected in parallel as detectors. The transglucosylation products were eluted using distilled water at a flow rate of 0.35 mL/min. For the structural isomer analysis, the HPLC system was used with a Unison UK-Amino (Imtact, Japan) column at 50 °C and a NQAD detector. The transglucosylation products were separated with a linear gradient under the following conditions; initial ratio of acetonitrile/water at 88 % and a flow rate of 0.4 mL/min, then decreased to 81 % for 30 min, then held at 81 % for 35 min, then decreased to 60 %, then held at 60 % for 10 min, and then increased back to 88 %. The fractionation of transglucosylation products were carried out using HPLC under the following conditions. The HPLC system was used with a Bio-Gel P2 (Bio-Rad) column at 60 °C and a refractive index detector. The transglucosylation products were eluted using distilled water at a flow rate of 9 mL/min.
Bio gel p 2
Manufactured by Bio-Rad
Sourced in United States
Bio-Gel P-2 is a gel filtration chromatography media. It is used for the separation and purification of macromolecules, such as proteins and peptides, based on their size and molecular weight. The porous structure of the gel beads allows smaller molecules to penetrate the pores, while larger molecules are excluded, resulting in their separation.
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Lab products found in correlation
Bio rex 70, by Bio-Rad (3 mentions)
Sephadex g 50, by GE Healthcare (3 mentions)
Tetrahydrofuran, by Merck Group (2 mentions)
Ammonium formate, by Merck Group (2 mentions)
1.2 μm syringefilter, by Avantor (2 mentions)
Hitrap q column, by GE Healthcare (2 mentions)
Hitrap, by Cytiva (2 mentions)
Mci gel ck04s column, by Mitsubishi (1 mentions)
Hi di formamide, by Thermo Fisher Scientific (1 mentions)
Abi 3130xl dna sequencer, by Thermo Fisher Scientific (1 mentions)
Pngase enzyme, by New England Biolabs (1 mentions)
Biogel p10, by Bio-Rad (1 mentions)
Lactobacilli mrs broth, by BD (1 mentions)
96 well plate, by Thermo Fisher Scientific (1 mentions)
Biophen anti xa, by Hyphen Biomed (1 mentions)
C18 silica gel, by Waters Corporation (1 mentions)
Prepfcfraction collector, by Gilson (1 mentions)
Reveleris x2, by Büchi (1 mentions)
Activated charcoal, by Fujifilm (1 mentions)
Bovine submaxillary mucin, by Merck Group (1 mentions)
52 protocols using bio gel p 2
1
Transglucosylation Product Analysis via HPLC
2
Astragalus mongholicus Bioactivity Evaluation
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Wild-simulated AR was obtained from Shanxi Hunyuan and identified by Professor Qin Xuemei of Shanxi University as the dry root of Astragalus mongholicus Bunge (harvested in 2017, growth period of 5 years). The medicinal material samples were kept in the sample bank of the Modern Research Center of Traditional Chinese Medicine of Shanxi University. Polyacrylamide gel Bio-Gel-P-2 was purchased from Bio-Rad (USA). Endo α-D-1,4-glucanohydrolase was obtained from Beijing Soleibao Technology Co., Ltd. Standard oligosaccharide samples were obtained from Glycarbo (Japan), and standard monosaccharide samples were purchased from Shanxi Jiujiu Trading Co., Ltd. The ELISA kit for mouse immunoglobulin G and cell counting kit (CCK-8) were acquired from Soleibao Technology Co., Ltd. Mouse monocyte macrophage Raw 264.7 and mouse lymphoma cells (YAC-1) were obtained from ATCC (American Type Culture Collection). Male BALB/c mice were purchased from Weitong Lihua Laboratory Animal Technology Co., Ltd. Phosphate buffered saline solution, lipopolysaccharide, trypsin, concanavalin A, and neutral red were purchased from Soleibao Technology Co., Ltd. Fetal bovine serum, DMEM high-glucose media and RPMI 1640 media were obtained from Soleibao Technology Co., Ltd.
3
N-Glycan Profiling from Purified IgG
4
Semi-purification of Mussel Toxins
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Mussels (Mytilus galloprovincialis) fed with the PST-producing dinoflagellate Alexandrium pacificum (strain ATHK) in the laboratory were extracted as described in Qiu et al [8 (link)]. A flow diagram on the semipurification of M-toxins is shown in Figure S1 . Extracts were lyophilized, dissolved in 0.1 M AcOH and then applied to 1.5 cm ID × 115 cm or 170 cm Bio-Gel P-2 (45–90 μm, Bio-Rad, Hercules, CA, USA) columns eluted with 0.1 M AcOH at a flow rate of 0.25 mL min-1. Fractions (1 mL) were collected with an automatic fraction collector and analyzed by LC-MS. Fractions containing M1, M3, M5 and M9 were combined and/or re-fractionated as above, as required. Fractions containing predominantly M2, M4, M6 and decarbamoyl M-toxins were lyophilized and dissolved in 10 mM ammonium bicarbonate (NH4HCO3), applied to a 1.5 cm ID × 170 cm Bio-Gel P2 column and eluted with 10 mM NH4HCO3 as above. Fractions from all isolation steps were combined into five separate solutions based on their M-toxin composition, as shown in Figure S2 . Solution 6 contained predominantly dcM6 isolated as part of a previous project [7 ]. All solutions obtained were lyophilized and re-dissolved in 0.1 M AcOH prior to analysis.
5
Purification of Enoxaparin Oligosaccharides
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Enoxaparins (80 mg of each), dissolved
in 0.8 mL of distilled water, were applied to a column (2.0 m x 1.0
cm) of Bio-Gel P-10 (90–180 μm mesh, BioRad, Herculs,
CA, USA), eluted with a 10% ethanol:1.0 M NaCl solution (1:1, v/v),
at a flow rate of 4 mL h–1. Fractions were collected
each 15 min and checked for absorbance at 232 nm. The fractions containing
the oligosaccharides were pooled, lyophilized, and dissolved in distilled
water. Thereafter, the fractions were desalting using a Bio-Gel P-2
(90–180 μm mesh, BioRad, 30 cm × 1 cm).7 (link)
in 0.8 mL of distilled water, were applied to a column (2.0 m x 1.0
cm) of Bio-Gel P-10 (90–180 μm mesh, BioRad, Herculs,
CA, USA), eluted with a 10% ethanol:1.0 M NaCl solution (1:1, v/v),
at a flow rate of 4 mL h–1. Fractions were collected
each 15 min and checked for absorbance at 232 nm. The fractions containing
the oligosaccharides were pooled, lyophilized, and dissolved in distilled
water. Thereafter, the fractions were desalting using a Bio-Gel P-2
(90–180 μm mesh, BioRad, 30 cm × 1 cm).7 (link)
6
Purification and Characterization of Bacterial Sugars
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Spent media at the stationary phase (A600 nm of ∼1.0) of B. thetaiotaomicron ΔBT1017 grown on CEAJ (50 ml) were centrifuged twice at 4000 × g for 6 min. The presence of sugars in the supernatants was confirmed by TLC as described above. Supernatants were filtered through a 0.2-μm syringe cap filter (PALL Life Sciences) and concentrated by freeze-drying using a CHRIST Gefriertrocknung ALPHA 1-2 freeze-dryer (Helmholtz-Zentrum Berlin) at −50°C to reduce the sample volume (to 20 ml). Sugars in supernatant were separated on a Bio-Gel P2 (Bio-Rad) size-exclusion system equilibrated in 5 mm acetic acid at a flow rate 0.6 ml min−1. Fractions (8 ml) were collected and analyzed by TLC. Fractions of interest were pooled and concentrated by freeze-drying and stored at room temperature until use.
7
Oligosaccharide Purification from Intestinal Mucins
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The intestinal mucins were submitted to β-elimination under reductive conditions (0.1 M KOH, 1 M KBH4 for 24 h at 45°C). The mixture of oligosaccharides alditols was purified by size exclusion chromatography on a column of Bio-Gel P2 (85 cm × 2 cm ID, 400 mesh, Bio-Rad, Richmond, CA, USA) equilibrated and eluted with water (10 mL/h) at room temperature. The oligosaccharide fractions, detected by UV absorption at 206 nm, were pooled for structural analysis.
8
Purification of Biological Toxins
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Each organ (muscles, gonads and liver) was homogenized separately with 1 volume of 1% acetic acid solution. Each homogenate was centrifuged at 4500 rpm for 15 min and the supernatant was collected and washed twice with the same volume of 1% acetic acid. Each combined extract was concentrated separately under reduced pressure and defatted with CH2Cl2. The aqueous layer obtained was concentrated, adjusted to pH 4~5 with 1 N NaOH solution, and then treated with activated charcoal. The adsorbed toxin was eluted with 3 volumes of 1% acetic acid in 20% ethanol and the eluted sample was combined and evaporated under vacuum pump. Further purification was carried out in according to the methods of Kotaki et al. (16 (link)), by gel filtration using a column (2.4 × 120 cm2) packed with Bio-Gel P-2 (Bio-Rad Laboratories, Richmond, CA, USA). Elution of the toxin was carried out with 0.03 N acetic acid at a flow rate of 60 mL/hr; then the toxicity of collecting fractions was determined by the bioassay.
9
Purification of CCK-oligosaccharides from L. lactis
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CCK-oligosaccharides were purified by the protocol described in our previous study [6 (link)]. Briefly, L. lactis CCK940 was cultured in Lactobacilli MRS broth (BD, Franklin Lakes, NJ, USA) at 30 °C for 20 h. The culture was inoculated to LM media [50 (link)], supplemented with 9.6% (w/v) sucrose and 7.4% (w/v) maltose, and incubated at 30 °C for 9 h. The culture supernatants were concentrated and separated by gel permeation chromatography (GPC; Bio-gel P2; Bio-Rad, Hercules, CA, USA). The oligosaccharide fractions were collected and lyophilized (SunilEyela, Seongnam, Korea) to determine their immunological effects.
10
Heparin Quantification Assay Protocol
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Unless otherwise noted, solvents and reagents
were purchased and directly used without further purification. Chemical
reagents were purchased from J&K Scientific Ltd. and TCI Shanghai
(Shanghai, China). Avidin beads and protamine were purchased from
Sangon Biotech (Shanghai, China). Bio-Gel P-2, P-4, or P-6 (45–90
μm) was purchased from Bio-Rad Laboratories (Hercules, California,
USA). C18 silica gel was purchased from Waters Corporation (Milford,
Massachusetts, USA). A Biophen anti-Xa (two-stage heparin assay) kit
was purchased from HYPHEN BioMed (Neuville-sur-Oise, France). 96-well
plates were purchased from Thermo Fisher Scientific (Waltham, Massachusetts,
USA).
were purchased and directly used without further purification. Chemical
reagents were purchased from J&K Scientific Ltd. and TCI Shanghai
(Shanghai, China). Avidin beads and protamine were purchased from
Sangon Biotech (Shanghai, China). Bio-Gel P-2, P-4, or P-6 (45–90
μm) was purchased from Bio-Rad Laboratories (Hercules, California,
USA). C18 silica gel was purchased from Waters Corporation (Milford,
Massachusetts, USA). A Biophen anti-Xa (two-stage heparin assay) kit
was purchased from HYPHEN BioMed (Neuville-sur-Oise, France). 96-well
plates were purchased from Thermo Fisher Scientific (Waltham, Massachusetts,
USA).
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