Gel electrophoresis was performed using 4%–12% or 12% Bolt Bis–Tris Plus gels (Thermo Scientific). Proteins were transferred to 0.45-μm polyvinylidene fluoride membranes for 60 min at 35 V. Proteins were crosslinked to the membrane via 0.4% (v/v) paraformaldehyde incubation in PBS for 30 min at room temperature, with rocking. The membranes were blocked for 60 min at room temperature in blocking buffer (5% [w/v] skim milk in 1× TBST (TBS and 0.05% [v/v] Tween-20)) and then incubated overnight at 4 °C with primary antibody directed against amino acids 15–123 of the α-synuclein protein (1:10,000 dilution, ref: 610786, BD Biosciences, Franklin Lakes, NJ) [8 (link), 24 (link)], diluted in the blocking buffer. The membranes were then washed three times with TBST and then incubated, for 60 min at room temperature, with horseradish peroxidase-conjugated secondary antibodies (ref: 172-1011, Bio-Rad, Hercules, CA) diluted 1:10,000 in the blocking buffer. Following another three washes with TBST, immunoblots were developed using Western Lightning enhanced chemiluminescence Pro (PerkinElmer, Waltham, MA) and imaged using X-ray film or the LiCor Odyssey Fc system.
Western lightning enhanced chemiluminescence pro
Manufactured by PerkinElmer
Sourced in United States
The Western Lightning Enhanced Chemiluminescence Pro is a reagent kit designed for the detection and analysis of proteins using the Western blot technique. The kit provides a sensitive and reliable solution for chemiluminescent protein detection, allowing for the visualization and quantification of target proteins.
Automatically generated - may contain errors
3 protocols using western lightning enhanced chemiluminescence pro
1
Western Blot Analysis of α-Synuclein
2
Western Blot Analysis of Signaling Proteins
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Following treatments or transfection, cells were washed with PBS and lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 120 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.1% Triton) in the presence of phosphatase and protease inhibitors on ice for 30 minutes. Cell debris was pelleted by centrifugation for 10 min at high speed. Protein concentration was quantified using a micro bicinchoninic acid protein assay kit (Thermo Fisher Scientific Inc). Proteins from control and treated cells were separated by Sodium dodecyl sulfate (SDS)–polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were electrotransferred to polyvinylidene difluoride membranes, which were then blocked overnight at 4°C with 5% non-fat dry milk in Tris-buffered saline (150 mM NaCl, 20 mM Tris–HCl, pH 7.5) containing 0.3% Tween-20 (TBST). Membranes were further washed in TBST and incubated with primary antibodies directed against either turboGFP (1/10,000), AKT, phosphorylated AKT (1/1,000), ERK, phosphorylated ERK (1/2,000), or anti-cavin-3 (1/1,500). Washing was then performed in TBST, followed by a 1 hour incubation with horseradish peroxidase-conjugated anti-rabbit IgG (1/10,000) or anti-mouse IgG (1/5,000) in TBST containing 5% non-fat dry milk. Immunoreactive material was visualized by Western Lightning Enhanced Chemiluminescence Pro (Perkin Elmer).
3
Western Blot Analysis of Signaling Pathways
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Following treatments or transfection, HT1080 cells were washed with phosphate-buffered saline and lysed with lysis buffer (50 mM Tris-HCl, pH 7.4, 120 mM NaCl, 5 mM EDTA, 0.5% Nonidet P-40, 0.1% Triton) in the presence of phosphatase and protease inhibitors on ice for 30 minutes. Cell debris was pelleted by centrifugation for 10 minutes at high speed. Protein concentration was quantified using a micro bicinchoninic acid protein assay kit (Thermo Fisher Scientific Inc., Waltham, MA, USA). Proteins from control and treated cells were separated by SDS-polyacrylamide gel electrophoresis (PAGE). After electrophoresis, proteins were electrotransferred to polyvinylidene difluoride membranes, which were then blocked overnight at 4°C with 5% nonfat dry milk in Tris-buffered saline (150 mM NaCl, 20 mM Tris-HCl, pH 7.5) containing 0.3% Tween-20 (TBST). Membranes were further washed in TBST and incubated with primary antibodies directed against MT1-MMP (1/10 000), p105, phosphorylated p105, Erk, phosphorylated Erk, IκB, phosphorylated IκB (1/1000), or GAPDH (1/1500). Washing was then performed in TBST, followed by a 1-hour incubation with horseradish peroxidase–conjugated anti-rabbit IgG (1/10 000) or anti-mouse IgG (1/5000) in TBST containing 5% nonfat dry milk. Immunoreactive material was visualized by Western Lightning Enhanced Chemiluminescence Pro (Perkin Elmer, Waltham, MA, USA).
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