The involvement of intact protein in immunomodulation was investigated upon heat-inactivation (hiMcES) in water bath at 100°C for 15 min. Mock-treated ES were kept 15 min at room temperature (Mock hi). To test the carbohydrates involvement, sodium metaperiodate-mediated modification of glycan moieties was performed. Briefly, 0.5 mg/ml of ES mixture was treated with 100 mM (vol/vol) of sodium acetate (pH 5.5) at room temperature. The tube content was divided to obtain test sample with addition of sodium metaperiodate (10mM) in sodium acetate buffer (McESΔCHO) or control mock-treated ES products (Mock ΔCHO) treated with the equivalent amount of sodium acetate buffer without sodium metaperiodate. The samples were incubated in the dark at room temperature with gentle shaking for 1h. Desalting and buffer exchange to PBS was accomplished using the Amicon Ultra-0.5 (3K MWCO; Merck Millipore) as per manufacturer instructions. To selectively precipitate proteins, ES products were saturated with ammonium sulfate up to a concentration of 80% (McESΔCHO). The precipitated proteins were obtained by centrifugation (6500g, 20 min) and dissolved in 100μl PBS and buffer-exchanged using Amicon-Ultra 0.5 (3K MWCO; Merck Millipore) as per manufacturer instructions.
Amicon ultra 0
Manufactured by Merck Group
Sourced in Germany, United States, Ireland
The Amicon Ultra-0.5 is a centrifugal filter unit designed for the concentration and purification of macromolecules. It features a regenerated cellulose membrane with a specific molecular weight cutoff, enabling the separation and concentration of molecules based on their size.
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Lab products found in correlation
Piercetm bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Pkh26, by Merck Group (3 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (2 mentions)
Arginase activity assay kit, by Merck Group (2 mentions)
Empower 3, by Waters Corporation (1 mentions)
Acquity uplc, by Waters Corporation (1 mentions)
Accq fluor reagent, by Waters Corporation (1 mentions)
Empower software, by Waters Corporation (1 mentions)
Rapid stain cbb kit, by Nacalai Tesque (1 mentions)
Jem 2010, by JEOL (1 mentions)
Specord 250 plus, by Analytik Jena (1 mentions)
Amicon ultra centrifugal filter, by Merck Group (1 mentions)
Pbs buffer, by Merck Group (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Streptavidin agarose beads, by Merck Group (1 mentions)
Genequant pro, by GE Healthcare (1 mentions)
Agilent 1290 infinity device, by Agilent Technologies (1 mentions)
G1316c fluorescence detector, by Agilent Technologies (1 mentions)
105 protocols using amicon ultra 0
1
Glycan Modulation Impacts Protein Immunoactivity
2
Enzymatic Preparation of AGF
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AGF was prepared by treating AF with β-galactosidase from Escherichia coli. 10 mg AF was dissolved in 2 mL of 50 mM Tris-HCl buffer (pH 7.2) containing 10 mM magnesium chloride, 5 mM β-mercaptoethanol and 200 U β-galactosidase. The reaction was allowed to proceed for 48 h at 37 oC, and then AGF (MW 42 kDa) was separated from β-galactosidase (MW 540 kDa, four equal subunits of 135 kDa) by ultrafiltration (Amicon Ultra-0.5, 100 k MWCO, Millipore) and purified by ultrafiltration (Amicon Ultra-0.5, 30 k MWCO, Millipore) against ultrapure water.
3
Encapsulation Efficiency of Phenolic Compounds
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Encapsulation efficiency (EE) was determined through the separation of the loaded particles from free phenolic extract. The separation was carried out using Amicon Ultra-0.5 (Amicon Ultra-0.5 mL 3 K device, Millipore Corp., Ireland), followed by centrifugation at 14000g for 30 min. EE was detemined according to Eq. ( 1):
where tpc corresponds to the total phenolic compounds and fpc corresponds to the free phenolic compounds.
where tpc corresponds to the total phenolic compounds and fpc corresponds to the free phenolic compounds.
4
SDS-PAGE Concentration and Analysis
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The 25 μg/mL samples were concentrated to 0.5 mg/mL using a centrifugal filter device (Amicon Ultra-0.5 with 3 kDa nominal molecular weight limit, Merck Millipore, Darmstadt, Germany), after which they were mixed with SDS sample buffer and analysed by SDS-PAGE or SDS-PAGE immunoblot assay as previously described [16 (link)]. PPS was used in the SDS-PAGE immunoblot assay.
5
Fluorescent Antibody Labeling Protocol
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Detection antibody labelled with biotin was coupled with CF-labelled streptavidin at 1:10 molar ratios at room temperature in the dark for 3 h. Unoccupied sites on streptavidin were blocked with excess dPEG4-biotin acid (10199; Quanta BioDesign, Ltd., Powell, OH, USA). Unconjugated streptavidin and dPEG4-biotin were removed by ultrafiltration (Amicon Ultra-0.5, 100 kDa; Merck Millipore, Billerica, MA, USA). The detection media contained prepared CF-labelled detection antibody (30 nM), BSA (2.5% [w/v]), and the indicated combination of the following additives (with the final concentrations): SYTOX (0.8 μM), glycine (5 mM), LPS (1 μg/mL), and/or ATP (5 mM).
6
Quantification of Free Amino Acids in Fermented Oat Drinks
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Fermented oat drink samples were centrifuged at 14,000×g for 20 min at room temperature (Hettich ROTANTA 460 R, fixed angle rotator). The supernatant was filtered through a 3 kDa molecular weight cut-off filter (Amicon® Ultra-0.5, Merck KGaA, Germany) and diluted with 2 parts of ultrapure water before analysis. Prior to injection, free amino acids were derivatized with AccQ•Fluor Reagent (Waters Corp., MA, USA) according to the manufacturer's procedure. Analysis of free amino acids was performed on an ultra-performance liquid chromatography (UPLC) system (Acquity UPLC; Waters Corp., MA, USA), including a binary solvent manager, a sample manager, and a photodiode array detector (PDA), controlled by Waters Empower™ 3.0 software (Build 3471, Waters Corp., MA, USA). Separations were performed on Waters Acquity UPLC AccQ•Tag Ultra Column (2.1 × 100 mm, 1.7 μm particle size) operated at +55 °C. The injection volume was 1.5 μL, the amino acids were eluted at a flow rate of 0.3 mL/min, and absorbance was recorded at 260 nm. The running time was 25 min. Empower software (Waters Corp., MA, USA) was used for data processing.
7
Purification and Microinjection of Anti-pStat3 Antibody
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Anti-phospho Stat3 (Tyr705) rabbit mAb (#9145, Cell Signaling Technology, Danvers, MA, USA) and isotype control rabbit mAb IgG (#3900, CST) were purified and concentrated with PBS using Amicon Ultra-0.5 (50 kDa, Merck). We subjected small aliquots of purified antibody and IgG to polyacrylamide gel electrophoresis, stained them using Rapid Stain CBB Kit (Nacalai Tesque), and determined the concentration as being 0.4 μg/μL. Purified anti-pStat3 antibody, control IgG, and PBS were microinjected into oocytes within 1 h after GVBD in TYH-HEPES, and the oocytes were then cultured in TYH-FCS.
8
Characterization of Lipid Nanoformulation FF-10832
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The morphology and size of FF-10832 were assessed using transmission electron microscopy (cryo-TEM, JEM-2010, JEOL Ltd., Tokyo, Japan) and dynamic light scattering (DLS, ELSZ-2000ZS, Otsuka Electronics, Tokyo, Japan), respectively. Stability was checked according to the guidelines recommended by the International Federation of Pharmaceutical Manufacturers & Associations (22 ), under storage conditions of 5°C ± 3°C for up to 24 months. The encapsulation efficiency (%) was calculated as follows:
where Ctotal and Cfree represent the concentrations of total and unencapsulated GEM, respectively. To prepare samples for measuring unencapsulated GEM, 200 μL of FF-10832 containing approximately 0.5 mg/mL of GEM were loaded onto an ultrafilter (Amicon® Ultra-0.5, MWCO: 10 K, Merck Millipore, MA) and centrifuged (7400×g, 20°C, 30 min). The concentrations of GEM and total lipids were analyzed using high-performance liquid chromatography (HPLC) and HPLC/charged aerosol detector (HPLC-CAD), respectively. The details of the procedures are provided in the Supplementary Methods. Experiments at each time point were performed in triplicate.
where Ctotal and Cfree represent the concentrations of total and unencapsulated GEM, respectively. To prepare samples for measuring unencapsulated GEM, 200 μL of FF-10832 containing approximately 0.5 mg/mL of GEM were loaded onto an ultrafilter (Amicon® Ultra-0.5, MWCO: 10 K, Merck Millipore, MA) and centrifuged (7400×g, 20°C, 30 min). The concentrations of GEM and total lipids were analyzed using high-performance liquid chromatography (HPLC) and HPLC/charged aerosol detector (HPLC-CAD), respectively. The details of the procedures are provided in the Supplementary Methods. Experiments at each time point were performed in triplicate.
9
Doxycycline Stability and Binding Kinetics
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Doxycycline was incubated at 1, 10, and 100 µM in 500 µL of DMEM medium added to HEK293-ACE2 cells seeded on 96-well plates (as in the transduction assay, see above). Stability and the bovine serum albumin (BSA)-bound fraction of doxycycline was assessed after 5 min and 0.5, 1, 2, 4, 6, and 24 h of incubation at 37 °C in humidified 5% CO2 (each well, in duplicate, corresponded to a different incubation time). At each timepoint, medium was removed, and an aliquot was used for determination of the doxycycline concentration.
Doxycycline binding to BSA was assessed only for 10 µM concentration. BSA-bound and free doxycycline were separated by ultrafiltration using Amicon Ultra-0.5 centrifugal filter devices (Merck Millipore, Vimodrone, Milano, Italy) with a MW cutoff of 30 KDa. Doxycycline in the three fractions (total, unbound, and BSA-bound) was measured using a validated HPLC-MS/MS method [13 (link)]. The amounts in the unbound and BSA-bound fractions were calculated using a mass balance approach to minimize inaccuracy due to confounding factors (e.g., non-specific binding of doxycycline to the filter membrane) [24 (link)].
Doxycycline binding to BSA was assessed only for 10 µM concentration. BSA-bound and free doxycycline were separated by ultrafiltration using Amicon Ultra-0.5 centrifugal filter devices (Merck Millipore, Vimodrone, Milano, Italy) with a MW cutoff of 30 KDa. Doxycycline in the three fractions (total, unbound, and BSA-bound) was measured using a validated HPLC-MS/MS method [13 (link)]. The amounts in the unbound and BSA-bound fractions were calculated using a mass balance approach to minimize inaccuracy due to confounding factors (e.g., non-specific binding of doxycycline to the filter membrane) [24 (link)].
10
Protein Fractionation using Centrifugal Filters
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Centrifugal filter devices (Amicon®- Ultra−0.5, Merck Millipore, Burlington, MA, USA) in five different “cut-off” sizes (3 K, 10 K, 30 K, 50 K, 100 K; K = 1000 Da) were filled with 500 µL sample and centrifuged for 15 min at 14,000× g at RT. The collection tube (filtrate) contained proteins smaller than the molecular cut-off. Proteins larger than the cut-off remained in the filter device after centrifugation (supernatant). To recover the sample in the filter device, the device was placed upside down in an empty tube and centrifuged for two minutes at 1000× g at RT.
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