Cultured rat hippocampal neurons at DIV14–16 were lysed with 1% triton X-100 lysis buffer with 1% seine/threonine phosphatase inhibitor (Sigma) and protease inhibitor cocktail (Sigma). Lysates were centrifuged at 14,000 g at 4 °C for 20 min after sonication. Supernatants were collected and protein concentration was measured using BCA assay kit. Equal amounts of protein were loaded on to polyacrylamide gels. Gels were transferred to PVDF membranes (Pall Life Sciences, Ann Arbor, MI), then the membranes were incubated with 10% BSA/PBS or 5% SKIM milk/PBS for 30 min at RT. After washing in TBST, PVDF membranes were incubated with the primary antibody for overnight at 4 °C, followed by the horseradish peroxidase (HRP)-conjugated secondary antibody (Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at RT. ECL solution (AbClon) and LAS 4000 (GE healthcare) were used to detect immunoreaction. Band intensities were calculated using imageJ.
Pvdf membranes
Manufactured by Jackson ImmunoResearch
Sourced in Panama
PVDF (Polyvinylidene fluoride) membranes are a type of laboratory equipment used for various applications in biochemical and molecular biology research. These membranes are made of a hydrophobic polymer material that provides a stable and durable surface for the immobilization and transfer of biomolecules, such as proteins, nucleic acids, and other macromolecules. PVDF membranes are commonly used in techniques like Western blotting, dot blotting, and immunodetection, where they serve as a support matrix for the separation and analysis of these biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor cocktail, by Merck Group (2 mentions)
Pvdf membrane, by Pall Corporation (2 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
Seine threonine phosphatase inhibitor, by Merck Group (1 mentions)
Las4000, by GE Healthcare (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
β actin, by Merck Group (1 mentions)
Gel logic 2200 imaging system, by Kodak (1 mentions)
Protein a sepharose beads, by GE Healthcare (1 mentions)
Bca assay kit, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by GE Healthcare (1 mentions)
Sds loading buffer, by Beyotime (1 mentions)
Rabbit anti actin, by Cell Signaling Technology (1 mentions)
Ecl reagent, by Share-Bio (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
4 protocols using pvdf membranes
1
Quantitative Western Blot Analysis of Hippocampal Neurons
2
Western Blotting Analysis of MAPK Signaling
3
Western Blot Analysis of Cultured Neurons
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Cultured neurons were lysed with 1% triton X-100 lysis buffer (20 mM Tris-HCl, pH 8, 1% triton X-100, 10% glycerol, 137 mM NaCl, 2 mM EDTA, 1 mM PMSF, 10 mM leupeptin, 1.5 mM pepstatin, and 1 mM aprotinin). Lysates were centrifuged at 14,000×g for 20 min at 4 °C, the supernatants were collected and, following protein determination by the BCA assay kit (Thermo Scientific, MA), samples were processed by SDS-PAGE and transferred to PVDF membranes (Pall Life Sciences, Ann Arbor, MI). PVDF membranes were blocked with 5% skim milk in PBS and then incubated with primary antibodies (1:2000) followed by horseradish peroxidase (HRP)-conjugated secondary antibodies (1:5000) (Jackson Immuno Research Laboratories, West Grove, PA) which were detected by ECL (enhanced chemiluminescence reagent (AbClon, Seoul, South Korea)). For immunoprecipitation, lysates (500 μg) were incubated for 90 min at 4 °C with primary antibodies (1:500) and then, after a wash, for one additional hour at 4 °C with Protein A-Sepharose beads (GE healthcare). Beads were washed by lysis buffer, eluted with sample buffer and boiling (5 min), and the eluates were processed for SDS-PAGE and western blotting.
4
Western Blot Analysis of SEMA6B in HEK-293T Cells
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HEK-293T cells were collected and lysed with a RIPA lysis buffer (Beyotime Biotechnology, China) 48 h after transient transfection. Total cell lysates were mixed with a 5 × SDS loading buffer (Beyotime Biotechnology, China), boiled for 10 min. Subsequently, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses were performed according to the standard protocols using 8% polyacrylamide gels. Proteins were transferred onto PVDF membranes (GE Healthcare, Germany). After blocking with 5% skim milk, PVDF membranes were incubated with primary antibodies overnight at 4°C, followed by incubation with HRP-conjugated antirabbit or antigoat (Jackson, United States) secondary antibodies at room temperature for 1 h. Blots were developed using the ECL reagent (Share-bio, China). All experiments were done in triplicates after optimal working conditions were determined. Primary antibodies used were rabbit anti-Actin (Cell signaling technology; 1:5000) and goat anti-SEMA6B (R&D Systems, Germany; 1:1000).
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