Ethyl acetate (EtOAc; J.T. Baker and Fisher Scientific) and Optima-grade methanol (MeOH; Fisher Scientific) were used. P. aeruginosa PAO1 wild type or ΔpqsA cells were grown overnight in LB (RPI) at 37°C with 200 rpm shaking. Bacterial cells were centrifuged at 5,000 rpm (Biofuge pico, Heraeus) and washed with 0.5% chelex treated CAA. New cultures were seeded in 0.5% chelex treated CAA at a starting OD600 of 0.05 and incubated at 37°C with 200 rpm shaking. After 48 h the cultures were centrifuged at 3,800 rpm (Eppendorf Centrifuge 5810 R) for 30 min at room temperature. The supernatant was decanted and filtered using a Nalgene Rapid-flow Filter Unit (0.2 µm aPES membrane; Thermo Scientific). The cell free supernatant was acidified to pH 1.8–2 with 6 M HCl. Pyochelin and pyochelin methyl ester were extracted with 3 volumes EtOAc and evaporated to dryness in a Rotavapor (Büchi; Rotavapor R-300) or a SpeedVac vacuum concentrator (SPD131DDA SpeedVac Concentrator; Thermo Scientific). Dried samples were resuspended in MeOH and stored at – 20° C. Samples were centrifuged for 5 min at 10,000 rpm (Thermo; Sorvall ST 40R) to remove nonsoluble particulates and diluted as needed in MeOH containing 1 µM glycocholic acid.
Nalgene rapid flow filter unit
Manufactured by Thermo Fisher Scientific
The Nalgene Rapid-flow Filter Unit is a laboratory filtration device used to filter liquids. It features a sturdy polycarbonate housing and a 47 mm diameter filter membrane.
Automatically generated - may contain errors
Lab products found in correlation
Fetal bovine serum (fbs), by Thermo Fisher Scientific (2 mentions)
Formic acid, by Thermo Fisher Scientific (2 mentions)
Fused silica capillaries, by Molex (2 mentions)
Lc ms grade water, by Thermo Fisher Scientific (2 mentions)
Acrylamide, by Thermo Fisher Scientific (2 mentions)
St 40r, by Thermo Fisher Scientific (1 mentions)
Optima grade methanol, by Thermo Fisher Scientific (1 mentions)
Spd131dda speedvac concentrator, by Thermo Fisher Scientific (1 mentions)
Rotavapor r 300, by Büchi (1 mentions)
Centrifuge 5810r, by Eppendorf (1 mentions)
Ebioscience cell stimulation cocktail, by Thermo Fisher Scientific (1 mentions)
Rpmi 1640 medium, by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Acetonitrile, by Thermo Fisher Scientific (1 mentions)
Methanol, by Thermo Fisher Scientific (1 mentions)
Hplc grade acetic acid, by Thermo Fisher Scientific (1 mentions)
3 trimethoxysilyl propyl methacrylate, by Merck Group (1 mentions)
Iodoacetamide, by Merck Group (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
6 protocols using nalgene rapid flow filter unit
1
Extraction and Analysis of Pyochelin Compounds
2
Purification and Characterization of CpaA-His Variants
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CpaA-His and its point mutation variants were purified from the supernatant of A. nosocomialis M2 (8 (link)). Briefly, A. nosocomialis M2 ΔcpaAB::frt strains carrying the pWH-based plasmids were grown in LB to mid-log phase. CpaA-His-tagged variants were purified by nickel affinity chromatography from cell-free filtered spent medium, as described before (8 (link)). Briefly, cells were pelleted at 8,000 × g for 10 min. Cell-free supernatants were obtained by filtration using a Nalgene Rapid-Flow filter unit (pore size, 0.2 μm) (Thermo Scientific), followed by concentration by approximately 3-fold using an Amicon Ultra-15 centrifugal filter unit (nominal molecular weight limit [NMWL], 10,000). Binding buffer (10×) was added to the cell-free supernatant prior to loading onto a nickel-nitrilotriacetic acid (Ni-NTA) agarose column (Gold Bio, St. Louis, MO) equilibrated with 1× binding buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole; pH 8.0). The column was washed with 20 column volumes (CV) of binding buffer and 10 CV of sashing buffer (50 mM NaH2PO4, 300 mM NaCl, 50 mM imidazole; pH 8.0). Proteins were eluted with elution buffer (50 mM NaH2PO4, 300 mM NaCl, 300 mM imidazole; pH 8.0). The purified proteins were concentrated, and buffer was exchanged for 20 mM HEPES, 150 mM NaCl, 50% glycerol (pH 7.4) using Amicon Ultra centrifugal filter units.
3
B Cell Response to Cigarette Smoke
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PBMCs or B cells (2 × 105 cells) were seeded in a 96-well plate in RPMI 1640 medium (Gibco) supplemented with 10% Fetal Bovine Serum (Thermo Fisher Scientific), 100 U/mL penicillin and 100 µg/mL streptomycin (both from Merck). Cells were stimulated for 48 h with recombinant human CD40L (1 µg/mL, R&D systems) and TLR9 agonist (0.77 µg/mL, ODN2006-TLR9 agonist CpG Class B, Invivogen) with addition of eBioscience™ Cell Stimulation Cocktail (plus protein transport inhibitors, Thermo Fisher Scientific) during the final 4 h of stimulation.
During the 48 h stimulation period, magnetically sorted B cells were cultured in 5% and 10% cigarette smoke extract (CSE). Cigarette smoke extract was obtained by passing the smoke of 10 cigarettes through 30 mL cell culture medium. After sterile filtering (Nalgene rapid-flow filter unit—Thermo Fisher Scientific) of the stock solution, a 1/20 or 1/10 dilution resulted in a final concentration of 5% or 10% CSE respectively. To neutralize BAFF upon in vitro CSE exposure, recombinant human BAFFR-Fc chimera protein (500 ng/mL, R&D systems) was added during the 48 h stimulation period.
During the 48 h stimulation period, magnetically sorted B cells were cultured in 5% and 10% cigarette smoke extract (CSE). Cigarette smoke extract was obtained by passing the smoke of 10 cigarettes through 30 mL cell culture medium. After sterile filtering (Nalgene rapid-flow filter unit—Thermo Fisher Scientific) of the stock solution, a 1/20 or 1/10 dilution resulted in a final concentration of 5% or 10% CSE respectively. To neutralize BAFF upon in vitro CSE exposure, recombinant human BAFFR-Fc chimera protein (500 ng/mL, R&D systems) was added during the 48 h stimulation period.
4
Acrylamide Proteomic Sample Preparation
5
Purification of Podo447 Antibodies from HEK293 Cells
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Example 19
Podo447 antibodies were expressed and secreted from HEK293 cells. HEK293 cells were transfected using 293-Free transfection reagent according to manufacture protocol (Novagen, Cat#: 72181). Cells were grown at 37° C. and 5% CO2 while shaking at 120 rpm for 120 hours. Antibodies in media were obtained by pelleting the cells at 2500 xg for 15 minutes at 4° C. The media was then filtered through Nalgene® Rapid-Flow™ Filter Units (Thermo Scientific, Cat#: 73520-984) and stored at 4° C. until purification.
Podo447 (rabbit or humanized) was purified using AKTA Pure FPLC system (GE Healthcare Life Sciences). The filtered media was passed through the HiTrap Mab Select SuRe column (GE Healthcare Life Sciences, Cat#: 11-0034-94). The column was then washed with 10 column volume (CV) of PBS, pH 7.4 and followed by elution with 20 CV gradient of 0.1 M glycine, pH 3.0. Each eluted fractions (1-mL) were mixed with 40 μL of 1 M Tris, pH 11. The eluted antibodies were concentrated and buffer-exchanged into PBS by Amicon Ultra-15 Centrifugal Filters with 30 k MWCO (Millipore, Cat#: UFC803024). The final product was filtered with a Costar Spin-X Centrifuge Tube, 0.22 μm Pore CA Membrane (Corning, Cat#: 8160) and its concentration was determined by A280 absorbance using the predicted extinction coefficient. The purity of the antibody was tested by SDS-PAGE, UPLC-SEC, and LC-MS.
6
Proteomic Sample Preparation Protocol
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Acrylamide was purchased from Acros Organics (NJ, USA). Ammonium bicarbonate (NH4HCO3), urea, dithiothreitol (DTT), iodoacetamide (IAA) and 3-(Trimethoxysilyl) propyl methacrylate, Tris-HCl, Hepes, MgCl2, phosphatase inhibitors: NaF, β-Glycerophosphate 2Na∙5H2O, Na-Orthovanadate, Na-Pyrophosphate∙10H2O and protease inhibitors: Antipain∙2HCl, Bestatin, Chymostatin, E-64, Leupeptin (hemisulfate), P-ramidon∙2Na, AEBSF, Aprotinin were purchased from Sigma-Aldrich (St. Louis, MO). LC/MS grade water, acetonitrile (ACN), methanol, and formic acid were purchased from Fisher Scientific (Pittsburgh, PA). Aqueous mixtures were filtered with Nalgene Rapid-Flow Filter units (Thermo Scientific) with 0.2 μm CN membrane and 50 mm diameter. Fused silica capillaries (50 μm i.d./360 μm o.d.) were obtained from Polymicro Technologies (Phoenix, AZ). Complete, mini protease inhibitor cocktail (provided in EASYpacks) was bought from Roche (Indianapolis, IN).
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