The G-/F-actin ratio was determined using a commercial G-/F-actin in vivo assay kit strictly following the manufacturer’s protocol (BKO37; Cytoskeleton). For immunoblotting, samples were subjected to SDS-PAGE and transferred onto nitrocellulose membranes (Amersham). After blocking with 5% nonfat dried milk powder and 0.05% Tween-20 in PBS, blots were incubated overnight with the appropriate antibodies. After several washes, blots were incubated for 30 min with secondary antibodies coupled to HRP. The signal was visualized with ECL chemiluminescence detection reagent (Thermo Fisher Scientific). Band intensities were quantified using ImageJ software, and results were expressed relative to the control condition.
Ecl chemiluminescence detection reagent
Manufactured by Thermo Fisher Scientific
Sourced in Canada, United Kingdom
The ECL chemiluminescence detection reagent is a laboratory product used for the detection and visualization of proteins in Western blot analysis. It utilizes a chemiluminescent reaction to produce light, which can be captured and quantified to measure the presence and abundance of target proteins.
Automatically generated - may contain errors
5 protocols using ecl chemiluminescence detection reagent
1
Determination of G-/F-actin Ratio
2
Western Blot Analysis of Protein Extracts
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Whole-cell lysates (WCLs) were prepared for electrophoresis by combining them 1:1 with 2× SDS buffer and boiling at 100ºC for 5–10 min.
IP and WCL samples were electrophoresed on 8–10% SDS–polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore) using a semidry transfer unit (Bio-Rad). Membranes were blocked in 5% BSA in Tris-buffered saline/Tween-20 (TBST) for at least 1 h before antibody incubation. Blots were washed three times for 8–10 min each in TBST after treatment with primary (16–72 h, 4ºC with nutation) and secondary (1 h, room temperature, with rocking) antibodies and visualized with ECL chemiluminescence detection reagent (Thermo Scientific, Ottawa, Canada) and film.
IP and WCL samples were electrophoresed on 8–10% SDS–polyacrylamide gels and transferred to polyvinylidene fluoride membranes (Millipore) using a semidry transfer unit (Bio-Rad). Membranes were blocked in 5% BSA in Tris-buffered saline/Tween-20 (TBST) for at least 1 h before antibody incubation. Blots were washed three times for 8–10 min each in TBST after treatment with primary (16–72 h, 4ºC with nutation) and secondary (1 h, room temperature, with rocking) antibodies and visualized with ECL chemiluminescence detection reagent (Thermo Scientific, Ottawa, Canada) and film.
3
Quantifying Protein Expression via Western Blot
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Leaf samples (0.2 g fresh weight) were ground in liquid nitrogen, extracted and quantified essentially as described by Harrison and Willingham [58 (link),59 (link)]. Total protein (8 µg) of each sample was loaded onto a 12% w/v SDS-PAGE gel, separated and transferred onto a nitrocellulose membrane. The resulting membranes were probed using FLAG antibodies (Sigma-Aldrich, Gillingham, UK) and detected by ECL chemiluminescence detection reagent (Thermo Scientific, Rockford, IL, USA) using horseradish peroxidase conjugated secondary antibody and visualized by chemiluminescence (PEQLAB Ltd., FUSION FX chemiluminescence detection system, Sarisbury Green, Fareham, UK).
4
Amyloid-Beta Oligomer Immunoblotting
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The AβO aliquots (1 μl) were spotted on a nitrocellulose membrane (Pierce). The membrane was blocked for 1 hour at room temperature with 10% nonfat milk in Tris-buffered saline containing 0.01% Tween 20 (TBS-T) and probed with 6E10 (1:10000 in 3% BSA), 4G8 (1:10000 in 3% BSA), OC (1:1000 in 5% nonfat milk). HRP-conjugated anti-mouse/rabbit IgG secondary antibodies (Abcam) were used at 1:5000 for 1 h at room temperature. Blots were detected using ECL chemiluminescence detection reagent (Thermo Fisher Scientific).
5
Protein Detection by Western Blotting
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Samples were subjected to SDS-PAGE and transferred onto Immobilon-P membranes (Millipore). After blocking with 5% non-fat dried milk powder and 0.05% Tween-20 in PBS, blots were incubated with the appropriate antibodies for 1 h. After several washings, blots were incubated for 30 min with secondary antibodies coupled to horseradish peroxidase. The signal was visualized with ECL chemiluminescence detection reagent (Thermo Scientific). Band intensities were quantified using Image J software and results were expressed relative to the control condition.
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