Milk was centrifuged at 2500 rpm for 20 min at 4 °C to remove the fat. The skimmed milk was acidified to pH 4.6 to precipitate casein and centrifuged at 100,000 × g at 20 °C for 1 h. The purification procedure was performed using an ÄKTA pure system (GE Healthcare, Uppsala, Sweden). First, after equilibration in a column with equilibration buffer (20 mM phosphate buffer (PB), pH 8.2), samples were loaded into a HiScreen SP Sepharose FF column (GE Healthcare, Uppsala, Sweden; 4.7 mL) and the protein was eluted with a linear gradient of 0–1 M NaCl in 20 mM PB, pH 8.2. Then, an Ultracel-30 membrane (Millipore Corporation, Billerica, MA, USA) was used to concentrate the fractions containing rhLZ on the ÄKTA Crossflow automated cross flow filtration system (GE Healthcare, Uppsala, Sweden) After purification, the rhLZ was exchanged by 20 mM PB and the quantity and quality of the purified rhLZ was detected by SDS-PAGE.
Ultracel 30 membrane
Manufactured by Merck Group
Sourced in United States
The Ultracel-30 membrane is a laboratory filtration device designed for the separation and concentration of macromolecules and particles. It features a high-performance polyethersulfone (PES) membrane with a molecular weight cutoff of 30 kDa. This membrane is suitable for various applications requiring sample preparation, purification, or concentration.
Automatically generated - may contain errors
Lab products found in correlation
Trypsin lys c, by Promega (3 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (3 mentions)
Kta crossflow automated cross flow filtration system, by GE Healthcare (2 mentions)
Kta pure system, by GE Healthcare (2 mentions)
Microcon 30 kda centrifugal filter unit, by Merck Group (2 mentions)
Polyethylenimine (pei), by Merck Group (2 mentions)
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Ultra 15 centrifugal filter unit, by Merck Group (2 mentions)
Glutathione agarose, by Merck Group (1 mentions)
Ultra 0.5 centrifugal filter unit, by Merck Group (1 mentions)
Affi gel protein a maps 2 kit, by Bio-Rad (1 mentions)
Amaxa cell line nucleofector kit r, by Lonza (1 mentions)
Tmt 6 plex labeling, by Thermo Fisher Scientific (1 mentions)
Transit lt1 transfection reagent, by Mirus Bio (1 mentions)
Human inflammatory cytokine kit, by BD (1 mentions)
Human tnf α elisa kit, by Thermo Fisher Scientific (1 mentions)
Cytokine standards, by BD (1 mentions)
Nativemark unstained protein standard, by Thermo Fisher Scientific (1 mentions)
Pierce silver stain kit, by Thermo Fisher Scientific (1 mentions)
Mini protean tgx precast gel, by Bio-Rad (1 mentions)
19 protocols using ultracel 30 membrane
1
Recombinant Human Lysozyme Purification
2
Purification of GST-Tagged Recombinant Proteins
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Expression was induced in 500 mL bacterial culture at OD595 = 0.8 by addition of 0.2 mM IPTG, followed by growth for additional 24 hr at 18°C. Cells were harvested by centrifugation and resuspended in lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 5% glycerol, 5 mM EDTA, 3 mM DTT, 0.1% Triton X-100, 200 μg/mL lysozyme, benzonase (10 units/mL), 10 μg/mL each of ‘CLAAPE’ protease inhibitors). The cells were then lysed by sonication. Cell extracts were clarified by centrifugation at 15,000 rpm at 4°C for 30 min. Cleared lysates were then incubated with glutathione-agarose (Sigma; G4510). glutathione-agarose with bound GST proteins was then placed in a column and washed with 20 bead-volumes of wash buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 100 mM KCl, 10% glycerol, 5 mM EDTA, 3 mM DTT, 0.1% Triton X-100). GST fusion proteins were then eluted with elution buffer (50 mM Tris-HCl pH 8.0, 300 mM NaCl, 10% glycerol, 2 mM EDTA, 3 mM DTT, 20 mM glutathione) in 1 mL fractions. Protein-rich fractions were pooled together and concentrated using Amicon Ultra-4 centrifugal filter unit with Ultracel®-30 membrane (Millipore; UFC803024).
3
Bioconjugation of Quantum Dots with Anti-HER2 Antibody
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A solution of QDs with a concentration of 0.5 mg/mL was prepared in phosphate-buffered saline and the anti-HER2 antibody was diluted in phosphate-buffered saline to a concentration of 100 μg/mL. Two different protocols were examined for bioconjugation based on the use of both EDC and NHS or EDC alone for activation of the QDs. For the first protocol, 1 mg of EDC powder and 1 mg of NHS powder were added to 0.5 mL of 0.5 mg/mL CdTe/CdSe/ZnSe-MUA QDs. For the second protocol, only 1 mg of EDC powder was added to the same concentration of QDs. To activate the QDs, the materials were incubated at room temperature in an orbital rotator for 40 minutes. After the activating process, all of the antibody solution (100 μg/mL) was added and left for one hour at room temperature on the orbital rotator, following which it was purified by centrifugal filtration using an Amicon Ultra-0.5 centrifugal filter unit with an Ultracel-30 membrane (Millipore, Billerica, MA, USA) for 10 minutes at a speed of 10 g. This removed all the unconjugated QDs and unreacted materials as a filtrate. The antibody-QD conjugate retained by the filtration membrane was diluted 50 times in phosphate-buffered saline and characterized using FTIR to confirm bioconjugation. Figure 1 is a schematic diagram that demonstrates the methodology used for bioconjugation and targeted localization of the surface receptors.
4
Purification of Fusion Proteins
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To obtain fusion proteins, expression experiments were conducted with the B. subtilis 1A751 cells harboring corresponding plasmids in flask as described above. The resulting culture medium was concentrated using an Amicon1 Ultra-15 centrifugal filter unit with Ultracel-30 membrane (Millipore, Billerica, MA) and changed to the enterokinase reaction buffer (50 mM Tris–HCl, 2 mM CaCl2, pH 7.6). To cleave the fusion protein, 1 mL of enterokinase (New England Biolabs Catalog # P8070S) was added to 20 mL of the twofold concentrate, and kept at 25 °C for 16 h. The resulting reaction mixtures were subjected to SDS–PAGE analysis and activity analysis.
5
Transient Transfection and Fc Fusion Protein Production
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COS-7 cells were transiently transfected or co-transfected with 5-10 µg of the indicated plasmids using the Amaxa Cell Line Nucleofector Kit R (Lonza) according to the manufacturer’s protocol. For co-transfection assays with HA-CD80 or empty pDisplay plasmids and GFP-expressing plasmids, 5 µg and 7.5 µg of the corresponding plasmids were used, respectively. For the production of Fc fusion proteins, HEK-293T cells were transiently transfected with 0.2 µg/cm2 of the indicated plasmid mixed with 6 µL/µg DNA of polyethylenimine (1mg/mL, Sigma-Aldrich) in 0.1 mL/cm2 of OPTIMEM medium (Gibco) for four hours. Then HEK cell cultures were washed, fresh medium added, and 6 days later Fc fusion proteins were collected from supernatants. Supernatants were further clarified by centrifugation and concentrated 30-fold using the Amicon Ultra-15 Centrifugal Filter Unit with an Ultracel-30 membrane (Millipore). Quantification of Fc fusion proteins was performed by sandwich ELISA using anti-Fc mAb and anti-human IgG-POD (Fc specific). For SPR analyses, Fc fusion proteins were purified from cell supernatants using Affi-Gel protein-A MAPS II kit (Bio-Rad).
6
Tandem Mass Tag Proteomics of PBMCs
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PBMC samples were transferred to a BSL-2 freezer and stored at − 80 °C until processed by iFASP [15 (link)]. Briefly, 30 µg of each inactivated PBMC lysate was added to 200 µL 8 M Urea/0.1 M Tris-HCl pH 8.5 and filtered through a Microcon-30 kDa Centrifugal Filter Unit with an Ultracel-30 membrane (Millipore; Cat# MRCF0R030) at 14,000×g for 15 min. Filters were washed 3× with 100 mM Tris pH 8.0, and proteins alkylated with 55 mM iodoacetamide followed by digestion with 4 µg Trypsin/Lys-C (Promega, Cat# V5071) overnight at 37 °C. TMT six-Plex labeling (Thermo Fisher Scientific; Cat# 90061) was performed directly on the FASP filters per the manufacturer’s instructions. TMT- labeled digests were then purified by C18 spin column, dried to completion by speed-vac and stored at − 20 °C until analyzed by LC–MS/MS.
7
Purification of Recombinant Human Lactoferrin
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The milk was centrifuged at 2500 rpm for 20 min at 4 °C to remove the fat. The skimmed milk was acidified to pH 4.6 to precipitate casein and was centrifuged at 100,000 × g at 20 °C for 1 h. The purification procedure was performed using an ÄKTA pure system (GE Healthcare, Uppsala, Sweden). First, after equilibrating three columns with equilibration buffer (20 mM phosphate buffer (PB), pH 7), the samples (100 L) were loaded onto a BPG 300/500 column, and the protein was eluted with 0.4 M NaCl and 1 M NaCl. Next, an Ultracel-30 membrane (Millipore Corporation, Billerica, MA, USA) was used to concentrate the fractions containing rhLF on an ÄKTA Crossflow automated cross-flow filtration system (GE Healthcare, Uppsala, Sweden). After purification, the rhLF was exchanged with 20 mM PB, and the quantity and quality of the purified rhLF were detected by SDS-PAGE.
8
Proteomic Analysis of Plasma Samples
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Plasma samples (6 time points/animal) were first processed in BSL-3 or BSL-4 containment by adding 25 µL SDS-PAGE solubilizing/reducing buffer to 75 µL sample and heating to 95 °C for 10 min. Samples were then removed from containment and stored at − 80 °C until processed by the iFASP method [28 (link)]. Briefly, 5 µL of each inactivated plasma sample was added to 200 µL 8 M Urea/100 mM Tris–HCL pH 8.5 (Solution UT8) and filtered through a Microcon-30 kDa Centrifugal Filter Unit with an Ultracel-30 membrane (Millipore, MRCF0R030) at 14,000 × G for 15 min. Following several washing steps with 100 mM Tris pH 8.0, proteins were alkylated with 55 mM Iodoacetamide and digested with 4 µg Trypsin/Lys-C (Promega, V5071) overnight at 37 °C. TMT 6-Plex labeling (Thermo Fisher, 90061) was performed directly on the FASP filters per the manufacturer’s instructions. All 6 single labelled samples were then combined at an equal volume, purified by C18 spin column, dried to completion by speed-vac and stored at − 20 °C until analyzed by LC MS/MS.
9
Isolation of pLV-tetO-Tfap2c Lentivirus
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To isolate pLV-tetO-Tfap2c lentivirus, HEK293T cells (cultured in DMEM with 10% FBS, 1mM NEAA and penicillin–streptomycin) were transfected with 500μl Opti-MEM containing 30 μl TransIT®-LT1 Transfection Reagent (Mirus, MIR2305), 10μg pLV-tetO-Tfap2c, 7.5μg psPAX2 and 2.5μg pMD2.G. Viral supernatants were collected at 48 and 72 hours, concentrated using Amicon Ultra-15 centrifugal filter units with an Ultracel-30 membrane (Millipore, Billerica, MA, USA), filtered through a 0.45 μM filter, and stored at −80°C until used.
10
Quantifying Cytokine Levels in Cell Cultures
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To analyze excreted TNFα levels, KBM-7-shBRCA2 or KBM-7-shLUC cells were treated with doxycycline (1 μg per mL) for 48 h. Proteins in supernatant culture media were concentrated using Microcon-30 kDa centrifugal filter units with Ultracel-30 membrane (Millipore). Subsequently, TNFα concentrations were determined using a human TNFα ELISA kit (KHC3011, Life Technologies).
IL-6, IL-8 and IL-10 levels were analyzed using the Human Inflammatory Cytokine Kit (BD Bioscience, #551811), according to the manufacturer’s protocol. In short, media were collected from BT-549 cells harboring different shRNAs, after treatment with doxycycline for 0, 2 or 4 days. Media samples (50 μL per sample) were incubated with IL-6, IL-8 and IL-10 capture beads for 3 h at room temperature. After two wash steps, samples were measured on an LSR-II (Becton Dickinson). Data were analyzed using FlowJo software, and cytokine concentrations were calculated using cytokine standards (BD Bioscience).
IL-6, IL-8 and IL-10 levels were analyzed using the Human Inflammatory Cytokine Kit (BD Bioscience, #551811), according to the manufacturer’s protocol. In short, media were collected from BT-549 cells harboring different shRNAs, after treatment with doxycycline for 0, 2 or 4 days. Media samples (50 μL per sample) were incubated with IL-6, IL-8 and IL-10 capture beads for 3 h at room temperature. After two wash steps, samples were measured on an LSR-II (Becton Dickinson). Data were analyzed using FlowJo software, and cytokine concentrations were calculated using cytokine standards (BD Bioscience).
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