HEK293 cells were transfected with a WT or mutant FLAG-IKKβ construct using Lipofectamine PLUS LTX reagent (Invitrogen) according to the manufacturer's instructions. Whole-cell lysates were then immunoprecipitated with anti-FLAG antibody and then incubated with 100 μM DMY-biotin. The immunoprecipitated FLAG-IKK-β-DMY-biotin ligands were separated by SDS-PAGE and transferred onto nitrocellulose membranes. After blocking with 1% BSA and washing with PBS-T [Tween-20 0.05%], the membranes were incubated with streptavidin horseradish peroxidase (Sigma) for 1 h and developed using ECL Western Blotting Detection Reagents (Invitrogen).
Ecl western blotting detection reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States, United Kingdom
ECL Western blotting detection reagents are a series of reagents used for the detection of proteins in Western blotting techniques. These reagents generate a chemiluminescent signal that can be captured and visualized to identify and quantify target proteins.
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Lab products found in correlation
Pvdf membrane, by Merck Group (8 mentions)
Pvdf membrane, by Bio-Rad (7 mentions)
Imagequant las 4000, by GE Healthcare (5 mentions)
Las 4000, by Cytiva (5 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (5 mentions)
Bradford assay, by Merck Group (4 mentions)
Ab6721, by Abcam (4 mentions)
Mtt powder, by Merck Group (4 mentions)
β actin, by Merck Group (4 mentions)
Gbox system, by Thermo Fisher Scientific (3 mentions)
Protein assay, by Bio-Rad (3 mentions)
Ne per nuclear and cytoplasmic extraction reagents kit, by Thermo Fisher Scientific (3 mentions)
Annexinv pi apoptosis assay kit, by BD (3 mentions)
Anti bcl 2, by Santa Cruz Biotechnology (3 mentions)
Anti iκbα, by Cell Signaling Technology (3 mentions)
Bcl2 associated x (bax), by Cell Signaling Technology (3 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (3 mentions)
Sybr premix ex taq, by Takara Bio (3 mentions)
Protran, by Cytiva (3 mentions)
β actin, by Abcam (3 mentions)
102 protocols using ecl western blotting detection reagent
1
Characterizing IKKβ Protein Interactions
2
Protein Extraction and Western Blot Analysis from Rat Hippocampus and BV-2 Cells
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Protein was extracted from the hippocampus of the rats or BV-2 cells by using RIPA buffer with an inhibitor of phosphorylase and protease. After extraction, the total protein content was measured using the BCA test. Protein samples were separated by SDS-PAGE and transferred to Polyvinylidene Fluoride (PVDF) membranes. Then, the membranes were blocked using nonfatty milk powder. The membranes were probed with the following primary antibodies: monoclonal rabbit anti-β-actin (Cat. No. 4967S), rabbit anti-GSK-3β (Cat. No. 9315S), rabbit anti-p-GSK-3β (Ser9) (Cat. No. 9336S), rabbit anti-p-Akt (S473) (Cat. No. 9271S), rabbit anti-Akt (Cat. No. 9272S), rabbit anti-PI3K p110α (Cat. No. 4249S, Cell Signaling), mouse anti-tau 46 (Cat. No. sc-32274), rabbit anti-p-tau (Thr205) (Cat. No. sc-101817) (Santa Cruz), rabbit anti-p-tau (Ser396) (Cat. No. ab109390), and rabbit anti-p-tau (Ser404) (Cat. No. ab92676) (Abcam), then incubated with a species-matched horseradish peroxidase-conjugated secondary antibody. The blots were visualized with ECL western blotting detection reagents (Invitrogen) and observed with Azure™ Biosystems c300. The optical density of immunoreactive bands was quantified using ImageJ.
3
Nuclei Lamin B1 Western Blot
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Nuclei lysates were prepared by direct lysis of 4 × 106 freshly defrosted nuclei in NuPAGE sample buffer, followed by 5 minutes sonication. The total protein concentration of the nuclei fraction was determined by Bradford Assay (Sigma) following the manufacturer’s instructions. Nuclei were heat-denaturated and resolved by SDS-PAGE on an 8% acrylamide gel. Proteins were transferred to 0.45 µm PVDF membrane using semi dry Power Blot Cassette (Thermo scientific). The membrane was blocked for 2 hours at room temperature with 5% (w/v) skimmed dried milk, 0.1% (v/v) TWEEN-20 in TBS and then probed against lamin B by incubation with the rabbit anti-Lamin B1 polyclonal antibody (Abcam) and subsequently a goat anti-rabbit antibody coupled to horseradish peroxidase (Abcam). The bands were visualised using the ECL Western Blotting Detection Reagents (Invitrogen) and the images were taken using Syngene GBox system. Images were processed in ImageJ.
4
Quantification of NDP52 Protein Levels
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Cell pellets from HeLa and MCF-7 cells, following NDP52 KD or control siRNA, were heat-denatured and resolved by SDS-PAGE. Membranes were probed against actin (Abcam, ab6276) and NDP52 by incubation with rabbit polyclonal primary antibody (1:2000 dilution, GeneTex, GTX115378) and, subsequently, a goat anti-rabbit antibody, coupled to horseradish peroxidase (1:15,000 dilution, Abcam, ab6721). Bands were visualised with ECL Western Blotting detection reagents (Invitrogen) using a ChemiDoc gel imager (Bio-Rad). For Ponceau S staining, membranes were incubated for 5 min in Ponceau S reagent (Sigma), washed three times with water and then imaged.
5
Protein Quantification and Western Blotting
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The total protein concentration was determined by Bradford Assay (Sigma) following the manufacturer’s instructions. Cell lysates were heat-denatured and resolved by SDS-PAGE. The membrane was probed against the endogenous proteins by incubation with primary Rabbit anti-myosin VI (1:500, Atlas-Sigma HPA0354863-100UL) or Mouse anti-RNAPII phospho Ser5 (1:500, Abcam Ab5408) and subsequently secondary Goat anti-rabbit antibody (1:15000 Abcam ab6721) or Goat anti-mouse antibody (1:15000, Abcam ab97023) coupled to horseradish peroxidase. The bands were visualised using the ECL Western Blotting Detection Reagents (Invitrogen) and the images were taken using Syngene GBox system. Images were processed in ImageJ.
6
Western Blot Protein Quantification
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After HYP treatment, the cells were lysed with RIPA. Protein concentrations were determined by Bio-Rad protein assay (Bio-Rad Laboratories, CA, USA). After electrophoretic separation, the gels were blotted and stained with primary antibodies. Binding of antibodies was visualized with peroxidase-coupled secondary antibodies using ECL Western Blotting detection reagents (Invitrogen, Scotland, UK). Band intensities were quantified using ImageJ software. Data were obtained from three independent experiments.
7
Protein Quantification and Western Blot Analysis
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The total protein concentration of the cytoplasmic and nuclei fraction was determined by Bradford Assay (Sigma) following the manufacturer’s instructions. Nuclei, cytoplasmic fractions and cell lysates were heat-denatured and resolved by SDS-PAGE. The membrane was probed against the endogenous proteins by incubation with the indicated rabbit polyclonal primary and subsequently a goat anti-rabbit antibody coupled to horseradish peroxidase (1:15,000 Abcam Ab6721). The following primary antibodies were used at the indicated dilutions: Rabbit anti-MVI (1: 500, Atlas-Sigma HPA0354863-100UL), Rabbit anti-NDP52 (1:2000, GeneTex GTX115378), Rabbit anti-alpha tubulin (1:1000, Santa Cruz sc5286), Rabbit anti-Lamin B (1:1000, Abcam ab16048 and rabbit anti-RNAP II phospho S5 (1:1000, Abcam Ab5131). The bands were visualised using the ECL Western Blotting Detection Reagents (Invitrogen) and the images were taken using Syngene GBox system. Images were processed in ImageJ. Uncropped blots are shown in Supplementary Figs. 9 and 10 .
8
Western Blot Procedure with ECL Detection
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The antibody‐based procedures used in this study comply with the recommendations made by the British Journal of Pharmacology. After drug treatment, adherent and floating cells were lysed with RIPA. Protein concentrations were determined using the Bio‐Rad protein assay (Bio‐Rad Laboratories, Inc., Hercules, CA, USA). After electrophoretic separation, gels were blotted and stained with primary antibodies. The binding of the antibody was visualized by peroxidase‐coupled secondary antibody using the ECL Western Blotting Detection Reagents (Invitrogen, Paisley, Scotland, UK). Band intensities were quantified by using the software ImageJ (NIH, Bethesda, MD, USA). Data were obtained from five independent experiments.
9
Western Blot Quantification Protocol
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The total protein concentration was determined by Bradford Assay (Sigma) following the manufacturer's instructions. Cell lysates were heat-denatured and resolved by SDS-PAGE. The membrane was probed against the endogenous proteins by incubation with primary Rabbit anti-myosin VI (1:500, Atlas-Sigma HPA0354863-100UL) or Mouse anti-RNAPII phospho Ser5 (1:500, Abcam Ab5408) and subsequently secondary Goat anti-rabbit antibody (1:15000 Abcam ab6721) or Goat anti-mouse antibody (1:15000, Abcam ab97023) coupled to horseradish peroxidase. The bands were visualised using the ECL Western Blotting Detection Reagents (Invitrogen) and the images were taken using Syngene GBox system. Images were processed in ImageJ.
10
Celastrol-Induced Apoptosis and Protein Analysis
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Apoptosis was detected by Annexin V staining kit (BD Biosciences, San Jose, CA, USA). In brief, cells were exposed to the indicated concentrations of celastrol for 24 h. Cells were then harvested and analyzed by flow cytometry using FITC-Annexin V and propidium iodide staining according to the manufacturer's instructions. Apoptotic cells were quantitatively counted by a flow cytometer (FACSAria III; BD Biosciences). Data acquisition and analysis were performed with the CellQuest (BD Biosciences) from triple independent experiments.
Protein extraction and western blotting. The celastrol treated cells were lysed with RIPA lysis buffer. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The cell lysates of samples were subjected to electrophoresis on SDS polyacrylamide gels and transferred to Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA), which were then blocked with 5% non-fat dry milk protein for 1 h. Membranes were then incubated with the indicated primary antibodies overnight at 4˚C. The binding of the antibody was visualized by peroxidase-coupled secondary antibody using the ECL Western Blotting detection reagents (Invitrogen). Band intensities were quantified by using the software ImageJ (NIH, Bethesda, MD, USA).
Protein extraction and western blotting. The celastrol treated cells were lysed with RIPA lysis buffer. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The cell lysates of samples were subjected to electrophoresis on SDS polyacrylamide gels and transferred to Hybond enhanced chemiluminescence nitrocellulose membranes (Amersham Biosciences, Piscataway, NJ, USA), which were then blocked with 5% non-fat dry milk protein for 1 h. Membranes were then incubated with the indicated primary antibodies overnight at 4˚C. The binding of the antibody was visualized by peroxidase-coupled secondary antibody using the ECL Western Blotting detection reagents (Invitrogen). Band intensities were quantified by using the software ImageJ (NIH, Bethesda, MD, USA).
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