To detect protein expression, TPC-1 cells that had been transfected for 24 h were washed in PBS and lysed with radioimmunoprecipitation assay buffer (50 mM TrisHCl pH 7.4; 150 mM NaCl; 2 mM EDTA; 1% NP-40; 0.1% SDS). The protein concentrations were determined using a bicinchoninic acid protein quantitation assay kit (Beyotime Institute of Biotechnology). Protein (20 µg) were separated by 10% SDS-PAGE and transferred onto a PVDF nylon membrane (Merck KGaA) at 4°C. Membranes were blocked for 30 min in 5% non-fat powdered milk at room temperature. After washing with TBS/Tween-20 (TBST) three times for 5 min each, the membranes were incubated with the anti-SPHK1 primary antibody (1:100; cat. no. ab71700; Abcam) for 8 h at 4°C. The membranes were then washed three times with TBST for 10 min each to remove free primary antibody. Mouse anti-GAPDH antibody (1:1,000 dilution; cat. no. ab8245; Abcam) was used as the loading control. After further washing, the membranes were incubated in a horseradish peroxidase-conjugated anti-rabbit IgG secondary antibody (1:1,000; cat. no. sc2004; Santa Cruz Biotechnology, Inc.) solution for 1 h at room temperature. All blots were visualized using an enhanced chemiluminescence solution (Merck KGaA) and analyzed using Quantity One Protein Analysis software version 4.6.7 (Bio-Rad Laboratories, Inc.).
Pvdf nylon membrane
Manufactured by Merck Group
The PVDF nylon membrane is a laboratory equipment product designed for filtration and separation applications. It is a microporous membrane made of polyvinylidene fluoride (PVDF) and nylon materials. The membrane provides a high level of chemical resistance and durability, making it suitable for use in a variety of laboratory environments.
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4 protocols using pvdf nylon membrane
1
Western Blot Analysis of SPHK1 Protein
2
Immunoblotting of Ovarian and Endometrial Cell Lines
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Immunoblotting assays of the various OVCAR3, CA5171, HEC1b and HEC151 transfectants were performed. Briefly, the cells were first lysed in the immunoprecipitation assay buffer, and the protein extracts were quantified using a BCA Protein Assay Kit (Pierce, Rockford, IL). Fifty μg of each cell lysate was then resolved by SDS/PAGE (12% gel), transferred onto a PVDF/nylon membrane (EMD Millipore, Billerica, MA), and probed with Abs specific to α-tubulin, YKL, Mcl-1, Bcl-2, Bak and Bax (Upstate Biotechnology, Lake Placid, NY). The membrane was then probed with horseradish peroxidase-conjugated secondary Ab (Upstate Biotechnology, Lake Placid, NY). The specific bands were visualized by an ECL® Western blotting system (GE Healthcare, Little Chalfont, UK). Protein levels were measured by densitometric analysis and normalized to the levels of α-tubulin (control) by ImageQuant 5.0 software (Molecular Dynamics Inc., Sunnyvale, CA). The expression level of each protein was presented as the fold change in comparison with the density of α-tubulin, and the expression levels in the original OVCAR3, CA5171, HEC1b and HEC151 cells were used as the reference.
3
Analyzing T Cell Signaling Pathways
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Sorted CD3+CD8+ T cells (1×106/well) were prepared with serum-free media and seeded in a 24-well plate for 6 hours. These T cells were treated with PBS and recombinant proteins, including IL-15, BTLA, and LIGHT (online supplemental table 2 ) for analyzing signaling pathways and harvested after 20 min of incubation. Twenty micrograms of each cell lysate were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis (SDS/PAGE) (10.0% gel), transferred onto a PVDF/nylon membrane (Millipore), and probed with various Abs (online supplemental table 3 ). The membrane was then probed with horseradish peroxidase (HRP)-conjugated secondary Ab. The specific bands were visualized with an ECL Western blot system (GE Healthcare).11 (link) To detect the expression of HVEM, PD-1, and GZMB in CD3+CD8+ T lymphocytes, IL-15, BTLA, and LIGHT (online supplemental table 2 ) were incubated with sorted T cells for 48 hours. LPS (0.5 µg/mL) and CpG-ODN (1 µg/mL) were incubated with sorted B cells for 24 and 48 hours to detect the expression of IL-15 and IL-15/IL-15Rα complex, respectively.
4
Immunoblotting Analysis of uPA Signaling
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Immunoblotting was used to assess the protein expression of uPA in different experiments. Cells were lysed with protein lysis buffer (137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2, 1 mM CaCl2, 1% Nonidet P40, 10% glycerol, 1 mg/ml BSA, 20 mM Tris/HCl (pH 8.0) and 2 mM orthovanadate). The protein extracts were quantified using a BCA (bicinchoninic acid) protein assay kit (Pierce). Then 50 μg of each cell lysate was resolved by SDS/PAGE (12% gel), transferred to a PVDF/nylon membrane (Millipore), and probed with antibodies specific to uPA, β-actin, extracellular signal-regulated kinase (ERK), phospho-ERK, Akt, c-Jun N-terminal kinase (JNK), phospho-JNK, p38, phospho-p38 (Upstate Biotechnology) or phospho-Akt (Ser473, Chemicon International). The membrane was then probed with either horseradish peroxidase-conjugated goat anti-mouse or goat anti-rabbit antibodies. The specific bands were visualized by an enhanced chemiluminescence Western blotting system (GE Healthcare).
The detection of ERK, AKT and p-P38 phosphorylation in the HEC1B/shuPA c19 and HEC1B/shuPA c25 cells treated with uPA were performed. Briefly, the cells were seeded and serum-free medium was replaced overnight, followed by treatment with 20 nM of recombinant uPA (R&D Systems, Minneapolis, MN) for 1 h49 (link). The cells were lysed and immunoblotting analysis was performed as described earlier.
The detection of ERK, AKT and p-P38 phosphorylation in the HEC1B/shuPA c19 and HEC1B/shuPA c25 cells treated with uPA were performed. Briefly, the cells were seeded and serum-free medium was replaced overnight, followed by treatment with 20 nM of recombinant uPA (R&D Systems, Minneapolis, MN) for 1 h49 (link). The cells were lysed and immunoblotting analysis was performed as described earlier.
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