Purified CD19 B cells from mouse spleens were stimulated with 10 μg/mL lipopolysaccharide (LPS, Sigma, L2880) for 72 h at 5% CO2 and 37 °C. The total protein of the activated B cells was then extracted by RIPA lysis buffer (Beyotime, Shanghai, China, P0013B) containing phosphatase inhibitor (Beyotime, P1050) and protease inhibitor (Beyotime, P1011). The prepared protein was separated on 10% SDS-PAGE gels and transferred on polyvinylidene fluoride membranes (0.45 µm; Millipore, Burlington, MA, USA, IPVH00010). Antibodies specific to Btk (Cell Signaling Technology, Danvers, MA, USA, 8547S), pBtk (Cell Signaling Technology, 87141S), PLCγ2 (Cell Signaling Technology, 34264S), pPLCγ2 (Cell Signaling Technology, 3871S), ERK (Cell Signaling Technology, 4695S), pERK (Cell Signaling Technology, 4370S), NF-κB p65 (Cell Signaling Technology, 4764S), pNF-κB p65 (Cell Signaling Technology, 3033S), and GAPDH (Cell Signaling Technology, 8884S) were incubated with the membranes overnight at 4 °C. The membranes were incubated with secondary peroxidase-conjugated antibodies the next day at room temperature for 1 h. Chemiluminescent signals were detected by the ECL method (Beyotime, Shanghai, China, P0018FS). The relative expression of each band was calculated with Fiji software.
Polyvinylidene fluoride membranes 0.45 m
Manufactured by Merck Group
Sourced in United States
Polyvinylidene fluoride (PVDF) membranes with a pore size of 0.45 micrometers are a type of laboratory filtration equipment. These membranes are used for various filtration and separation processes in research and industrial applications.
Automatically generated - may contain errors
Lab products found in correlation
Ripa lysis buffer, by Beyotime (1 mentions)
P erk, by Cell Signaling Technology (1 mentions)
Nf κb p65, by Cell Signaling Technology (1 mentions)
Protease inhibitor, by Beyotime (1 mentions)
Phosphatase inhibitor, by Beyotime (1 mentions)
Lipopolysaccharides (lps), by Merck Group (1 mentions)
P1050, by Beyotime (1 mentions)
P btk, by Cell Signaling Technology (1 mentions)
Plcγ2, by Cell Signaling Technology (1 mentions)
P plcγ2, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
P nf κb p65, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Enhanced chemiluminescence reagent, by Bio-Rad (1 mentions)
3 protocols using polyvinylidene fluoride membranes 0.45 m
1
Western Blot Analysis of Activated B Cells
2
Western Blot Analysis of Neuroprotective Pathways
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Cell lysates (40 µg) were extracted after 24-hour OGD treatment, separated by electrophoresis (Bio-Rad, CA, USA) at 90 V, and transferred to polyvinylidene fluoride membranes (0.45 µm; Millipore, Boston, MA, USA) at 250 mA. The membranes were blocked and incubated at 4°C overnight with primary antibodies: rabbit monoclonal anti-MT2 (1:200, ImmunoClone, Houston, TX, USA); rabbit polyclonal anti-HIF-1α (1:1000), rabbit polyclonal anti-phosphorylated p38-mitogen-activated protein kinase (p-p38MAPK)/rabbit polyclonal anti-p38MAPK (1:1000), rabbit polyclonal anti-phosphorylated extracellular-signal-regulated protein kinase (p-ERK)/ rabbit polyclonal anti-ERK (1:1000; Cell Signaling Technologies); rabbit polyclonal anti-VEGF (1:1000), rabbit polyclonal anti-matrix metalloproteinase 9 (MMP-9; 1:1000), rabbit polyclonal anti-cyclin D1 (1:500) (Proteintech, Wuhan, China); and mouse monoclonal anti-β-actin (1:800, Boster). The membranes were washed and incubated in secondary antibodies (1:5000, Proteintech) at room temperature for 120 minutes and visualized by an enhanced chemiluminescence (ECL) system (Thermo), and analyzed by ImageJ software (NIH, Bethesda, MD, USA) (Zhong et al., 2017; He et al., 2018).
3
Western Blotting Quantitative Analysis
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Western blotting was performed using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by protein transfer to polyvinylidene fluoride membranes (0.45 µm, Millipore, Darmstadt, Germany). The membrane was washed three times and blocked by soaking the membrane in TBS-T buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, and 0.1% Tween 20) containing 5% bovine serum albumin (GenDEPOT) for 30 min. Then, the membrane was incubated with primary antibody (1:1000 dilution) overnight at 4 • C, washed again three times, and treated with secondary antibody (1:2000 dilution) for 1 h at RT. All primary (mouse monoclonal β-actin, rabbit monoclonal COX-2, rabbit monoclonal iNOS, rabbit monoclonal NF-κB p65, and rabbit monoclonal NF-κB pp65) and secondary antibodies (anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody, anti-mouse IgG, HRP-linked antibody) were purchased from Abcam (Cambridge, UK) and Cell Signaling Technology (Beverly, MA, USA) [30] . Protein expression was visualized using an enhanced chemiluminescence reagent (Bio-Rad, Hercules, CA, USA), following the manufacturer's instructions. Quantitative analysis was conducted using the free software ImageJ (version 1.52a for Windows; NIH, Rockville, MD, USA).
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