Total protein from the hippocampal tissue and BV2 microglial cells was prepared and extracted using the BCA Protein Extraction Kit (KGP2100, KeyGEN Biotechnology Co., Ltd, Jiangsu, China). Protein concentration was measured using the BCA Protein Assay Kit (KGP902, KeyGEN Biotechnology Co., Ltd, Jiangsu, China). Equal amounts of protein (50 µg per lane) were resolved on a 10% or 12% sodium dodecyl sulfate–polyacrylamide gel (SDS-PAGE), and then transferred onto 0.22-μm polyvinylidene fluoride (PVDF) membranes (Millipore, USA) blocked with 5% non-fat milk, and incubated with the following primary antibodies overnight at 4°C: rabbit anti-Bcl-2 (1:500), Bax (1:1000), cleaved caspase-3 (1:1,000), NLRP3 antibody (1:500), caspase-1 p20 (1:500), ASC (1:500), IL-18 (1:500), IL-1β (1:500), TNF-α (1:1,000), and β-actin (1:2,000). Membranes were washed with TBST (TBS containing 1‰ Tween 20) and subsequently incubated with Goat Anti-Rabbit IgG (H&L) Secondary antibody Conjugated (1:5,000). Detection was performed with an Odyssey Infrared Imaging System CLX-0796 (LI-COR, Lincoln, NE, USA) and quantified via densitometry with Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA). All experiments were performed in triplicate.
0.22 μm polyvinylidene fluoride pvdf membrane
Manufactured by Merck Group
Sourced in United States
The 0.22-μm polyvinylidene fluoride (PVDF) membranes are a type of filtration media used in various laboratory applications. These membranes have a pore size of 0.22 micrometers, which allows them to effectively retain particles, bacteria, and other small entities while permitting the passage of fluids.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imaging system clx 0796, by LI COR (2 mentions)
Bca protein extraction kit, by Keygen Biotech (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Bca protein assay kit, by Keygen Biotech (1 mentions)
Chemiluminescence kit, by Merck Group (1 mentions)
Bca protein analysis kit, by Merck Group (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Ecl plus western blotting detection reagent, by Bio-Rad (1 mentions)
Chemidoc xrs plus luminescent image analyzer, by Bio-Rad (1 mentions)
Protein extraction kit, by Keygen Biotech (1 mentions)
β actin, by Bioworld Technology (1 mentions)
Runx2, by Abcam (1 mentions)
Imagequant las 4000 system, by GE Healthcare (1 mentions)
Camkii, by Cell Signaling Technology (1 mentions)
Phosphor p65, by Cell Signaling Technology (1 mentions)
Phosphor iκbα, by Cell Signaling Technology (1 mentions)
Gsk3β, by Cell Signaling Technology (1 mentions)
β catenin, by Abcam (1 mentions)
5 protocols using 0.22 μm polyvinylidene fluoride pvdf membrane
1
Western Blot Analysis of Hippocampal and Microglial Proteins
2
Protein Extraction and Western Blot Analysis
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Cultured cells or tissues were homogenized with RIPA lysis buffer (Millipore) containing protease inhibitors for over 30 min. Nuclear protein lysates were generated using a nuclear protein extract kit (Servicebio) following the manufacturer’s instructions. The homogenates were centrifuged at 15,000 × g at 4 °C for 10 min and then the protein concentration was detected by a BCA protein analysis kit (Sigma). Approximately 20–40 μg of total protein solution was loaded onto SDS–polyacrylamide gels and transferred onto 0.22-μm polyvinylidene fluoride (PVDF) membranes (Millipore). The membranes were blocked with 5% bovine serum albumin for 30 min at room temperature and then probed with the indicated primary antibody at 4 °C overnight, followed by incubation with the horseradish peroxidase-conjugated secondary antibody at room temperature for 1 h. A chemiluminescence kit (Millipore) was used to detect the target bands. Information about the antibodies used is provided in Additional file 1 : Table S1.
3
Protein Extraction and Analysis from Human Peritoneal Mesothelial Cells
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Human peritoneal mesothelial cells were treated as mentioned above. Cells were washed twice with PBS and lysed in lysis buffer containing 50 mM Tris‐Cl [pH 7.4], 150 mM NaCl, 1% Triton X‐100, 1% deoxycholic phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 5.0 mM sodium pyrophosphate, 1.0 g/ml leupeptin, 0.1 mM phenylmethylsulfonyl floride and 1 mM DTT. Cells were clarified by centrifugation (13 000 g, 15 min., 4°C). Bicinchoninic acid (BCA) kit with Varioskan multimode microplates spectrophotometer from Thermo Fisher Scientific Inc. (Waltham, MA, USA) was used to detect the concentration of protein in supernatants.
For sample loading, equal amounts of protein (20–30 μg) were separated by 10%, 12% or 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto the 0.22 μm polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). The immune complexes were formed by incubation of proteins with primary antibodies overnight at 4°C followed by the appropriate HRP‐labelled secondary antibodies for 1 hr at 37°C. Detection of the immune blots was performed using the ECL plus Western blotting detection reagents (Bio‐Rad, Hercules, CA, USA) and the ChemiDoc XRS Plus luminescent image analyzer (Bio‐Rad)23 .
For sample loading, equal amounts of protein (20–30 μg) were separated by 10%, 12% or 15% sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) and transferred onto the 0.22 μm polyvinylidene fluoride (PVDF) membranes (Millipore, Boston, MA, USA). The immune complexes were formed by incubation of proteins with primary antibodies overnight at 4°C followed by the appropriate HRP‐labelled secondary antibodies for 1 hr at 37°C. Detection of the immune blots was performed using the ECL plus Western blotting detection reagents (Bio‐Rad, Hercules, CA, USA) and the ChemiDoc XRS Plus luminescent image analyzer (Bio‐Rad)
4
Western Blot Analysis of NF-κB Pathway
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The HUVECs were treated with SDG or DMSO (1%) for 1 h and then treated with LPS or PBS for 6 h. Total or nuclear proteins of colonic tissue or cells were prepared and extracted using the Protein Extraction Kit (KeyGEN Biotechnology Co., Ltd., Jiangsu, China). Then, the concentration of protein was measured using the BCA Protein Assay Kit (KGP902). 50 μg protein was resolved by a 10% sodium dodecyl sulfate- (SDS-) polyacrylamide gel (SDS-PAGE) to evaluate the expression of the protein and then transferred onto 0.22 μm polyvinylidene fluoride (PVDF) membrane (Millipore, USA). After blocking with 5% nonfat milk, the membrane incubated with primary antibodies overnight at 4°C as follows: rabbit anti-NF-κB p65 (1 : 1000), p-NF-κB p65 (1 : 1000), p-IκB-α (1 : 1000), IκB-α (1 : 1000), p-Akt (1 : 1000), β-actin (1 : 3000), and PCNA (1 : 1000). After washing for 3 × 5 min in PBST (contain 1‰ tween20), goat anti-rabbit IgG (H&L) secondary antibody conjugated (1 : 5000) was incubated for 1 h at RT. Then, results were detected using an Odyssey Infrared Imaging System CLX-0796 (LI-COR, Lincoln, NE, USA), and semiquantitative analysis was then performed using Image J software.
5
Evaluating Odonto/Osteogenic Protein Expression
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For evaluating the expression of odonto/osteognic proteins, nDPSCs and iDPSCs treated or untreated with baicalin or BMS345541 or DKK1 were harvested, washed and lysed. To analyze the expressions of NF-κB and β-catenin/Wnt pathway related proteins, the cytoplasm protein as well as nucleoprotein were extracted from iDPSCs treated with 20 µM baicalin for 0, 15, 30, and 60 min. After loading the protein onto a 10% SDS-PAGE gel for electrophoresis, electroblotted (Bio-Rad, USA) the protein onto 0.22 μm polyvinylidene fluoride (PVDF) membrane (Millipore, USA) at 300 mA for 1 h. After blocking membranes with solution at 25℃, incubated them with primary antibodies (DSP, Santa Cruz; RUNX2, Abcam; OSX, Abcam; OCN, Abcam; phosphor-P65; Cell Signaling, Boston, MA, USA, P65, Cell Signaling, Boston, MA, USA; phosphor IκBα, Cell Signaling; IκBα, Cell Signaling; β-catenin, Abcam; GSK-3β, Cell Signaling; NLK, Cell Signaling; CaMKII, Cell Signaling; β-ACTIN, Bioworld, Minneapolis, MN, USA; Histone, Bioworld) at 4 °C all night long. Membranes were then washed for 1 h with PBST blended with secondary antibodies (Boster, China) at 37 °C before visualized with ImageQuant LAS4000 system (GE Healthcare, USA). β-ACTIN and H3 were set as the internal parameters, respectively.
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