Cells were grounded into a homogenate in RIPA buffer (CWBIO) containing protease and phosphatase inhibitors (Thermo Fisher). The supernatants were lysed in ice for 30 min and collected by centrifuging at 12,000 rpm at 4 °C for 30 min. The total protein concentration was determined with the BCA kit (Beyotime). The supernatants were mixed with loading buffer (Beyotime), and equal amounts (40 μg) of protein were loaded onto sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). After the separated protein was transferred to the polyvinylidene difluoride (PVDF, Thermo Fisher) membrane, the PVDF membrane was blocked with 5% skim milk for 1 h and then incubated with the primary antibodies (Table 2 ) overnight. The PVDF membrane was then incubated with the secondary antibody for 1 h before applying the enhanced chemiluminescence (ECL) kit (Thermo Fisher) for visualization. Tris Buffered Saline with Tween® 20 (TBST) was used to wash the membranes thrice before each step. The results were analyzed using ImageJ software.
Polyvinylidene fluoride (pvdf)
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom, Germany, China
PVDF is a type of fluoropolymer material widely used in various laboratory applications. It is a durable, chemically resistant, and thermally stable polymer that exhibits excellent mechanical properties. PVDF is often utilized in the manufacturing of laboratory equipment, such as pipettes, valves, and tubing, due to its ability to withstand a wide range of chemicals and temperatures.
Automatically generated - may contain errors
Lab products found in correlation
Bovine serum albumin (bsa), by Merck Group (9 mentions)
Bca protein assay, by Thermo Fisher Scientific (8 mentions)
Ripa buffer, by Thermo Fisher Scientific (8 mentions)
Ecl kit, by Thermo Fisher Scientific (8 mentions)
Bca protein assay kit, by Beyotime (7 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (7 mentions)
Bca protein kit, by Thermo Fisher Scientific (7 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Carbon black, by Thermo Fisher Scientific (5 mentions)
Protease inhibitor, by Thermo Fisher Scientific (5 mentions)
Bca assay, by Thermo Fisher Scientific (5 mentions)
Sds page, by Bio-Rad (5 mentions)
Ab6721, by Abcam (5 mentions)
Bca kit, by Beyotime (5 mentions)
Protease and phosphatase inhibitor, by Thermo Fisher Scientific (5 mentions)
Hydrochloric acid (hcl), by Merck Group (4 mentions)
Bca assay, by Beyotime (4 mentions)
Ripa buffer, by Merck Group (4 mentions)
Gapdh, by Abcam (4 mentions)
β actin, by Santa Cruz Biotechnology (4 mentions)
222 protocols using polyvinylidene fluoride (pvdf)
1
Protein Extraction and Western Blot Analysis
2
Fabrication of PVDF-ZnO Nanorods Film
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Zinc oxide nanorods (ZnO NR) are grown on the PVDF film through the in situ method followed by hydrothermal process22 (link). Ionization of the PVDF film is carried out with Zn2+ through the addition of 0.4 g of zinc acetate (Zn(CH3COO)2·2H2O, Alfa Aesar) into 4 g of polyvinylidene fluoride (PVDF, Alfa Aesar) dissolved in 5:3 volume% of N-methyl-2-pyrrolidone:acetone solvents. The solution is probe sonicated at an amplitude of 35% for 1 h and then polymerized at 70 °C overnight. Later, the ionized PVDF film is treated hydrothermally with an equal molecular ratio of Zn(CH3COO)2·2H2O and hexamethylenetetramine (HMT, Alfa Aesar) for 12 h at 80 °C. Finally, the PVDF–ZnO NR film is washed with ethanol and water and dried at 70 °C.
3
Electrochemical Characterization of Anode Materials
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To characterize the electrochemical properties of the samples, coin-type half cells with the samples as anodes were prepared in an Ar-filled glove box. The slurry was prepared using a paste mixer with the as-prepared powder samples as anode active materials (70 wt%), Ketjen black as a conducting agent (Wellcos, 10 wt%), and polyvinylidene fluoride (10 wt% PVDF, Alfa Aesar, 20 wt%) as a binder. The as-prepared slurry was coated on Cu foil (Hohsen Corporation) using a doctor blade method and then dried in a 110 °C convection oven for 24 h. The half-cell was assembled with an active material-coated electrode, 1.1 M LiPF6 as an electrolyte, polyethylene (Wellcos) as a separator, and lithium metal as a counter electrode. Charge–discharge curves of the samples were obtained using a multichannel battery tester (WBCS3000L, Wonatech Co.) in the potential range of 0–3 V vs. Li/Li+ with various current densities of 100, 300, 500, 800 and 1600 mA g−1. Cyclic voltammograms (CVs) of the samples were measured at a scan rate of 0.02 mV s−1 in the potential range of 0–3 V vs. Li/Li+. Galvanostatic intermittent titration technique (GITT) measurement was performed at a current density of 100 mA g−1 for 10 min intervals, with rest periods of 60 min, in the potential range of 0–3 V Li/Li+.
4
Protein Extraction and Western Blot Analysis
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Proteins were extracted using 1 × RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA, USA) containing a phosphatase and protease inhibitor cocktail (Thermo Fisher Scientific). Western blotting was performed following standard procedures. Briefly, protein lysates were separated using 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene fluoride membranes (PVDF; Thermo Fisher Scientific). After blocking with blocking reagent (Thermos Fisher Scientific), the membranes were incubated overnight with primary antibodies at 4 °C. After incubation with secondary antibodies, the PVDF membranes were thoroughly washed and detected using electrochemiluminescence solution (Thermo Fisher Scientific). Primary antibodies (MEK1/2, P-MEK1/2, ERK1/2, P-ERK1/2, COX-2, P65, P-P65, IκBα, P-IκBα, and GAPDH) and secondary antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA) [26 (link)].
5
PVDF Membrane Functionalization Protocol
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Low fluorescence polyvinylidene membrane (PVDF) with a pore size of 0.45 was purchased from Bio-rad laboratories (California, USA). PVDF were cut to 1 × 1 cm dimensions and wetted in 100% methanol (Fisher scientific, Leicestershire, UK) followed by 3 washes in sterile MilliQ. Next PVDF membranes were coated with ligands, rhEpCAM (25 μg mL -1 ) or Protein A (30 μg mL -1 ) overnight at 4 °C. Next membranes were washed in Phosphate buffered saline (PBS), (EMD Milliepore, Milleroca, USA) and blocked in blocking buffer consisting of 3% BSA (Sigma Aldrich, St Louis, USA) in PBS for 1 h at RT. Finally, the PVDF membranes were washed once in PBS and incubated in cell culture medium prior to use.
6
Fabrication of LiMn1.5Ni0.5O4 Cathodes
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An 80:10:10 wt.% mixture of LiMn1.5Ni0.5O4, carbon black (super P conductive, 99+%, Alfa Aesar) and polymer binder poly(vinylidene fluoride) (Alfa Aesar) was used to prepare cathodes. The slurry of the mixture was spread on the Al-foil, and then it was dry-heated at 120 °C. The cathode discs/working electrodes were made after the coated foil was punched. The reference/counter electrode was Li metal (99.9% trace metal basis, Sigma-Aldrich) and LiPF6 (1 mol/L in a mixed solvent of ethylene carbonate, dimethyl carbonate, and diethyl carbonate with a volume ratio of 1:1:1, MTI Corporation) was used as an electrolyte in all the cells prepared. The CR2032 cells fabrication was carried out in an Ar-filled glove box.
7
Synthesis and Characterization of LiPF6/DMC Electrolytes
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The lithium hexafluorophosphate salt [LiPF6 (water content <10 ppm)] and dimethyl carbonate solvent [DMC (water content <100 ppm)] were all purchased from Guangdong Candlelight New Energy Technology Co., Ltd. Electrolytes of LiPF6 in DMC with different salt concentrations were prepared by dissolving 0.7596 g or 4.1775 g of LiPF6 salt in 5 mL DMC solvent and sonicating to prepare solutions of CCEs and HCEs. The salt-to-solvent molar ratios ranged from 1:1 to 5.5:1 and were denoted as 1 M LiPF6/DMC or 5.5 M LiPF6/DMC, respectively. The preparation was conducted in an argon glovebox with water and oxygen content below 0.1 ppm. The ionic conductivity and the viscosities of 1 M LiPF6/DMC and 5.5 M LiPF6/DMC are 3.97 mS cm-1, 1.69 mm2 s−1 and 3.80 mS cm−1, 54.45 mm2 s−1, respectively. Those electrodes were fabricated by mixing the active materials of LiNi0.5Mn1.5O4 (LNMO) (from Shenzhen Baxter New Energy Materials Co., Ltd.), acetylene black (Shenzhen Baxter New Energy Materials Co., Ltd.), and polyvinylidene fluoride (Alfa Aesar) binder with a mass ratio of 8:1:1 in N-methyl-2-pyrrolidone (NMP, Alfa Aesar). The resultant slurry was coated evenly on aluminum foil by a film applicator (150 μm thickness) and then dried under vacuum at 110°C for 12 h, which loading amount was around 1.2–2.0 mg cm−2 after drying.
8
Fabrication of Composite Materials
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Titanium, Titanium carbide, and aluminium (325 mesh size) were purchased from Materials Korea (Rep. of Korea). Graphene (M-25, average size of 25 μm) was obtained from Ditto Technology (Rep. of Korea). Ethanol (98%), agar, hydrochloric acid (HCl 35%), tetrahydro furan (THF), and aniline were supplied by Samchun (Seoul, Korea). Polyethylene terephthalate (PET) binder (fibre diameter 2.2 dtex, 5 mm) and carbon fibre (fibre diameter 7 μm, 6 mm length) were obtained from TORAY product, (Tokyo, Japan), while polyacrylamide (PAM) was purchased from Sigma Aldrich (Seoul, South Korea). Iron(iii ) chloride (FeCl3 97%), lithium fluoride (LiF 300 mesh), pyrrole (98%), thiophene (99%), trimesic acid (98%), 4-aminophenol (98%), acrylic acid, N-methyl-2-pyrrolidone (NMP), and polyacrylic acid (PAA) were obtained from Sigma Aldrich (Seoul, South Korea). Polyvinyl alcohol (PVA 89%) and polyvinylidene fluoride (PVDF) were supplied by Alfa Aesar (Seoul, Korea). Chitiooligosaccharide (10 000 MW) was provided by Sunchon National University, Biomedical polymer lab, (Jeonju Korea). Glycerin was obtained by reagent from Duksan (Seoul Korea). N,N-Dimethylformamide (DMF) was purchased from Daejung (Seoul Korea). All of the chemicals were used without further purification.
9
Synthesis of Magnetic Composites from Orange Peel
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Hydrated ferric (III) chloride (FeCl3·6H2O, > 98.0%), hydrated magnesium nitrate (Mg(NO3)2·6H2O, > 99.0%), sodium hydroxide (NaOH, analytical grade), hydrochloric acid (HCL, 37%, analytical grade), and potassium hydroxide (KOH, analytical grade) were obtained from Merck. Polyvinylidene fluoride (PVDF), N-Methyl-2-pyrrolidone (NMP), and carbon black were sourced from Alfa Aesar. Orange peels (OP) were collected from the farm in Damietta Governorate, Egypt, between October and December.
10
Western Blot Analysis of Protein Expression
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Transfected cells were collected and lysed with 2× Lysis Buffer (Sigma–Aldrich, St. Louis, Missouri, U.S.A.). Total proteins in the cell or tissue samples were quantitated using BCA protein assay kit (Pierce Biotechnology, Rockford, IL, U.S.A.) following the instructions. The obtained proteins were first separated using 10% SDS/PAGE and then transferred on to polyvinylidene fluoride membranes (PVDF, Invitrogen, Carlsbad, CA). The membranes were blocked using TBST solution which was supplemented with 5% skim milk at 4°C for 1 h, and then incubated with first antibodies against AKT3 (1:1000, CST#9272, Boston, U.S.A.), NFATc2 (1:500, Abcam ab2722, Cambridge, U.K.), PPP3CA (1:2000, Abcam ab3673, Cambridge, U.K.), FOS (1:200, Abcam ab7963, Cambridge, U.K.) and AKT1S1 (1:1000, CST#2691, Boston, U.S.A.) overnight at 4°C, followed by the incubation with second antibodies for 1.5 h at room temperature. The blots were visualized with enhanced chemiluminescence kit (GE Healthcare, Chalfont St. Giles, Buckinghamshire, U.K.) following the manufacturer’s protocols.
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