Cells were lysed with 1X Laemmli sample buffer (50 mM Tris-Cl (pH6.8), 100 mM DTT, 2% SDS, 12.5% glycerol, 0.1% Bromophenol blue), sonicated in a Diagenode Bioruptor (4°C, 30 s ON/15 s OFF cycles, 5 min) and boiled at 95°C for 15 min. Protein separation was performed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% 200:1 bis-acrylamide gels. Proteins were transferred onto nitrocellulose membranes and saturated in PBS-BSA 3% for 1 h. The membranes were incubated with the primary antibody at 1ug/mL in PBS-BSA 1% overnight, followed by 1 h incubation with the HRP-conjugated secondary antibody diluted 1/10,000 (mouse) or 1/20,000 (rabbit) times in PBS-Tween 0.05%. Detection of the chemo-luminescent signal was performed using the Amersham ECL Western Blot Detection (GE Healthcare) according to the manufacturer’s instructions. Images were acquired on a Bio-Rad ChemiDoc™ XRS+ System and analysed with the Image Lab software.
Amersham ecl western blot detection
Manufactured by GE Healthcare
The Amersham ECL Western Blot Detection system is a chemiluminescence-based detection method for protein analysis. It is used to visualize and quantify specific proteins in Western blot experiments.
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Western Blot Analysis of Protein Samples
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Western Blot Protein Analysis
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Cells were lysed with 1× Laemmli sample buffer (50 mM Tris-Cl at pH 6.8, 100 mM DTT, 2% SDS, 12.5% glycerol, 0.1% Bromophenol blue), sonicated in a Diagenode Bioruptor for 5 min (cycling 30 sec on/15 sec off) at 4°C, and boiled for 15 min at 95°C. Protein separation was performed by SDS–polyacrylamide gel electrophoresis (SDS-PAGE) using 12.5% 200:1 bis-acrylamide gels. Proteins were transferred onto nitrocellulose membranes and saturated in 3% PBS-BSA for 1 h. The membranes were incubated overnight with the primary antibody at 1 µg/mL in 1% PBS-BSA, followed by 1 h of incubation with the HRP-conjugated secondary antibody diluted 1/10,000 (mouse) or 1/20,000 (rabbit) in 0.05% PBS-Tween. Detection of the chemo-luminescent signal was performed using Amersham ECL Western blot detection (GE Healthcare) according to the manufacturer's instructions. Images were acquired on a Bio-Rad ChemiDoc XRS+ system and analyzed/quantified with Image Lab software. The antibodies used are shown in Table 4 .
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