Briefly, the total protein was extracted from the samples using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China). Protein concentrations were quantified using a Bicinchoninic Acid Protein Assay kit (Biosky Biotechnology, Nanjing, China) according to the manufacturer's protocol. Next, protein samples (30 µg/lane) were separated by SDS-PAGE on 10% gels and transferred to polyvinylidene difluoride membranes (0.45 µm; EMD Millipore, Billerica, MA, USA). Membranes were blocked for 2 h at room temperature with 1–5% BSA (Sangon Biotech Co., Ltd.) in TBS-Tween-20 [containing 5 mmol/l Tris-HCl (pH 7.6), 136 mmol/l NaCl and 0.05% Tween-20] and then incubated with the aforementioned antibodies against p65, p-p65, p50, p-p50, CD9, CD63 and tubulin overnight at 4°C. Subsequently, the membranes were incubated with secondary antibody [horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (IgG; cat. no. A0208) or anti-mouse IgG (cat. no. A0216); 1:5,000; Beyotime Institute of Biotechnology] at 37°C for 2 h. Signals were detected using enhanced chemiluminescence western detection reagents (Thermo Fisher Scientific, Inc.) and quantified using Image-Lab version 5.2.1 software (Bio-Rad Laboratories, Inc.).
Polyvinylidene difluoride membranes 0.45 m
Manufactured by Merck Group
Sourced in United States
Polyvinylidene difluoride (PVDF) membranes with a pore size of 0.45 µm are a type of laboratory filtration membrane. They are used for various applications in research and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescence western detection reagents, by Thermo Fisher Scientific (1 mentions)
Image lab version 5, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Primary antibodies against bdnf, by Abcam (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
β actin, by OriGene (1 mentions)
Imagequant las 4000 mini, by GE Healthcare (1 mentions)
Caspase 3, by Cell Signaling Technology (1 mentions)
Cyclin d1, by Cell Signaling Technology (1 mentions)
β actin, by GE Healthcare (1 mentions)
Pro prep protein extraction solution, by iNtRON Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
4 protocols using polyvinylidene difluoride membranes 0.45 m
1
Western Blot Analysis of Protein Expression
2
BDNF Protein Expression Analysis in Hippocampus
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The expression of BDNF protein in the hippocampus was evaluated by Western blot analysis. The concentrations of proteins extracted with TRizol reagent (Thermo Fisher Scientific, Inc., Tokyo, Japan) were determined using Bradford’s method with bovine serum albumin (BSA) as a standard. Each amount of proteins was subjected to SDS-PAGE and then transferred to polyvinylidene difluoride membranes (0.45 µm, Merck Millipore, Billerica, MA, USA). The membranes were blocked with 3% BSA in Tris-buffered saline (TBS) with 0.05% Tween-20 (TBST) for 1 h at room temperature, and incubated overnight at 4 °C with primary antibodies against BDNF (1:3000; Abcam, Cambridge, MA, USA) and tubulin (1:5000; Abcam). Next, the membrane was washed with TBST three times and incubated with secondary horseradish peroxidase-conjugated antibody (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h. The protein bands were visualized using the EzWestLumi plus kit (ATTO, Tokyo, Japan) and AE-9300 Ez-Capture (ATTO). The intensity of protein bands was analyzed using ImageJ software (NIH Image, Bethesda, MD, USA).
3
Protein Expression Analysis of MMP2 and NKCC1
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Peritumoral edema tissue was ground in liquid nitrogen and dissolved in a RIPA Lysis Buffer (Beyotime Institute of Biotechnology), which contained the protease inhibitor phenylmethanesulfonyl fluoride. The total protein was extracted from the tissue, and the protein concentration was determined using the BCA Protein Assay Kit (catalog no. P0012; Beyotime Institute of Biotechnology) and stored at −80°C for additional analysis. Equal amounts of protein (>20 µg) from each group were loaded and resolved using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (0.45 µm; EMD Millipore, Boston, MA, USA). The membranes were blocked with 5% non-fat milk in Tris-buffered saline, and subsequently incubated with rabbit anti-rat MMP2 and rabbit anti-rat NKCC1 antibodies at 4°C overnight. The membranes were incubated with goat anti-rabbit secondary antibody (monoclonal; dilution, 1:2,000; catalog no. TA130024; OriGene Technologies, Inc.) for 2 h at room temperature. The optical density of bands was determined using ImageJ software (version 1.48; National Institutes of Health, Bethesda, MA, USA) and the data were quantified by normalization to the density of β-actin (OriGene Technologies, Inc.).
4
Axitinib's Impact on Cyclin D1 and Caspase-3 Regulation
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CIPp and CIPm cells were seeded in 6-well plates and incubated overnight. Cells were treated with 0, 2, 4, or 8 µM axitinib for 24 h and harvested for protein extraction and quantification using PRO-PREP Protein Extraction Solution (iNtRON Biotechnology, Korea) and a Bio-Rad DC Protein Assay Kit (Bio-Rad Laboratories, USA), respectively, according to the manufacturers’ instructions. For analyzing cyclin D1 and caspase-3 expression, 25 µg protein sample was loaded and separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins were transferred onto polyvinylidene difluoride membranes (0.45 µM; Merck Millipore, USA) and blocked with 5% bovine serum albumin in Tris-buffered saline with Tween 20 for 1 h at room temperature. The membranes were probed using antibodies against cyclin D1 (1:1,000; Cell Signaling Technology, USA), caspase-3 (1:1,000; Cell Signaling Technology), and β-actin (1:1,000; Santa Cruz Biotechnology, USA) at 4°C overnight. The membranes were washed and treated with goat anti-mouse horseradish peroxidase-labeled secondary antibody (Bethyl Laboratories, USA) or goat anti-rabbit horseradish peroxidase-labeled secondary antibody (Enzo Life Sciences) for 1 h. Immunoreactive bands were detected using ImageQuant Las4000 mini (GE Healthcare Life Sciences, USA) and normalized with respect to the levels of the loading control β-actin.
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