Sciatic nerves from adult control littermates (n = 6) and SC-p75NTR-KO (n = 6) mice were dissociated in lysis buffer (2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 140 mM NaCl and 1% Triton X-100, pH 7.8, with protease inhibitors from Roche) and centrifuged at 10.000 × g for 10 min at 4°C. Total protein concentration was determined using the Bicinchoninic Acid kit from Sigma. Protein lysates were run on 12% SDS-PAGE (20 μg/lane) and electro-blotted for 1.5 h onto polyvinylidenedifluoride (PVDF) filters (Amersham) in 192 mM glycine, 25 mM Tris–HCl, pH 8.0. Membranes were then blocked and incubated overnight at 4°C with the primary antibodies: rabbit anti-p75NTR (1:500, Promega, Cat. #G323A) and mouse anti-β-actin (1:5000, Sigma, Cat. #A5441). Following a washing step, membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1:1000, swine anti-rabbit, Dako, Cat. # P0217; rabbit anti-mouse, Dako # P0260) and blots visualized with the Amersham ECL plus western blotting detection reagents (GE Healthcare) and Fuji film LAS1000. Densitometry was performed with QuantityOne software (Bio-Rad).
Pvdf filter
Manufactured by Cytiva
Sourced in United States
The PVDF filter is a type of filtration media made of polyvinylidene fluoride (PVDF) material. It is designed to provide high-efficiency filtration for a variety of applications in the laboratory setting. The core function of the PVDF filter is to effectively remove particulates, microorganisms, and other unwanted components from liquid samples, while maintaining the integrity of the desired components.
Automatically generated - may contain errors
Lab products found in correlation
Bicinchoninic acid kit, by Merck Group (1 mentions)
Amersham ecl plus western blotting detection reagent, by GE Healthcare (1 mentions)
Mouse anti β actin, by Merck Group (1 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Anti glut1, by Novus Biologicals (1 mentions)
Anti porin, by Santa Cruz Biotechnology (1 mentions)
Mitoprofile total oxphos human wb antibody cocktail, by Abcam (1 mentions)
Anti mct1, by Novus Biologicals (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
Reax 2, by Heidolph (1 mentions)
Zinc formalin, by Merck Group (1 mentions)
Nta software, by Malvern Panalytical (1 mentions)
Nanosight ns300, by Malvern Panalytical (1 mentions)
Suprapur, by Merck Group (1 mentions)
7 protocols using pvdf filter
1
Quantifying p75NTR Protein Expression
2
Protein Expression Analysis in CD4+ T Cells
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CD4+ T cell protein extracts (~ 30 μg) were boiled in Laemli sample buffer (Sigma-Aldrich) for 5 min, resolved on 10% SDS-polyacrylamide gels and transferred onto PVDF filters (Amersham). Membranes were blocked for 1 h in Tris-buffered saline (TBS), 0.05% Tween-20, 5% non-fat dry milk, followed by overnight incubation with specific primary antibodies diluted in the same buffer. The primary antibodies used are listed as follows: anti-GLUT1 (1:1000, Novus Biologicals), anti-MCT1 (1:1000, Novus Biologicals), anti-β-actin (1:8000, Sigma-Aldrich). Mitochondria-rich pellet (15 μg) was separated on a SDS-PAGE and, after blocking, membranes were incubated with MitoProfile total OXPHOS human WB antibody cocktail (1:1000, Abcam) and anti-Porin (1:1000, Santa Cruz). After washing with 0.1% Tween in TBS, membranes were incubated with a peroxidase-conjugated secondary antibody for 1 h, washed and developed using the ECL chemiluminescent detection system (Clarity™ Western ECL Substrate Biorad). The densitometric analyses of blots were performed by a computerized image processing system (Image J, 1.0 version).
3
Determining VD3 Solubility in PBS/Tween 80
4
Pharmaceutical Adsorption Experiments
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Batch adsorption experiments were performed by contacting the adsorbents (AAC or CAC) with solutions of pharmaceutical (CBZ, SMX or PAR) prepared either in ultrapure or in the collected wastewater. Pharmaceutical solutions of CBZ, SMX or PAR, with an initial concentration (C0) of 5 mg L -1 were shaken together with a known concentration (M) of the corresponding adsorbent in polypropylene tubes. The tubes were shaken in a head-over-head shaker (Heidolph, Reax 2) at 80 rpm, under controlled temperature (25.0 ± 0.1 ºC). After shaking, solutions were filtered through 0.2 µm PVDF filters (Whatman) and analysed for the residual concentration of pharmaceutical by micellar electrokinetic chromatography (MEKC) (as described in section 2.6).
Control experiments, i.e. the pharmaceutical solution in absence of adsorbent, were run in parallel. All experiments were run in triplicate.
Control experiments, i.e. the pharmaceutical solution in absence of adsorbent, were run in parallel. All experiments were run in triplicate.
5
Photocatalytic MB Degradation Assay
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The MB degradation test was carried out in a dark room to compare the photocatalytic efficiency of each sample. An aqueous solution of 5 mg/L was prepared using MB as a pollutant; in addition, 0.2 g of the TiO2 nanofiber sample and 20 mL of deionized water were added to a quartz beaker, and stirred for 30 min. After that, 200 mL of MB aqueous solution was added to the TiO2 dispersed water and stirred for 2 h in a dark room, with temperature control for mixing and stabilizing. After that, the distance between the UV lamp (6 W, 365 nm) and the quartz beaker was fixed at 10 cm, and the beaker was stirred at 240 rpm during the photodegradation reaction. The reaction was carried out for 3 h, and the mixed solution was sampled every 30 min using a syringe. The TiO2 photocatalyst in the sampled solution was filtered and removed using a syringe filter (PVDF filter, 0.2 μm, Whatman, Marlborough, MA, USA); in addition, the filtered solution was stored in a cuvette and refrigerated to block the incident light.
6
Nanoparticle Tracking Analysis of Extracellular Vesicles
7
Measuring Dissolved Copper in Microcosms
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To measure dissolved Cu concentrations, 30 mL of microcosm water was sampled before and after each water renewal, filtered (0.45 µm polyvinylidene difluoride (PVDF) filter, Whatman), acidified with 0.5% (v:v) nitric acid (Suprapur, Merck), and stored at 4 °C until analysis. Filtered water samples were analyzed using inductively coupled plasma -mass spectrometry (ICP-MS XSeries II, Thermo Electron). Quality controls were routinely checked using a certified reference material (Environment Canada, TM 27-3, Lake Ontario natural water) to check analytical accuracy (97%) and precision (± 12%).
In the control microcosms, dissolved Cu concentrations remained very low (0.4 ± 0.1 µg L-1) throughout the experiment. In all Cu microcosms, at 2 h after water renewal, mean dissolved Cu concentrations were 63.0 ± 8.8 µg L-1 with no significant difference among treatments. As previously observed in similar microcosm experiments (Lambert et al., 2012 (Lambert et al., , 2016)) , Cu concentrations decreased strongly between each water renewal (from 43% to 60% according to the sampling time). Mean dissolved Cu concentrations before water renewal were 32.2 ± 8.0 µg L-1, with no significant difference among treatments.
In the control microcosms, dissolved Cu concentrations remained very low (0.4 ± 0.1 µg L-1) throughout the experiment. In all Cu microcosms, at 2 h after water renewal, mean dissolved Cu concentrations were 63.0 ± 8.8 µg L-1 with no significant difference among treatments. As previously observed in similar microcosm experiments (Lambert et al., 2012 (Lambert et al., , 2016)) , Cu concentrations decreased strongly between each water renewal (from 43% to 60% according to the sampling time). Mean dissolved Cu concentrations before water renewal were 32.2 ± 8.0 µg L-1, with no significant difference among treatments.
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