Total proteins were harvested using the CelLytic Extraction kit (Roche, Basel, Switzerland) containing protease inhibitors and then quantified using the BCA Protein Assay Reagent kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. After separating proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis, protein was transferred to polyvinylidene fluoride membranes and then blocked in 5% defatted milk. Using the primary antibodies and anti-rabbit linked to horseradish peroxidase (1:5000) (Santa Cruz Biotechnology, Dallas, TX) as the second antibody, the target proteins were probed and then visualized using the ECL PlusWestern Blotting System (Thermo Fisher Scientific, Waltham, MA). β-actin was use as a loading control. The primary antibodies included the following: AKT (1:1000), phosphorylated AKT (pAKT)(Ser473) (1:500), GSK3β (1:500), pGSK3β(Ser9) (1:500), cyclin D1 (1:1000), p27Kip1 (1:1000), PI3K (1:1000), (Cell Signaling Technology, Boston, MA) and β-actin (1:2000) (Santa Cruz Biotechnology, Dallas, TX).
Ecl plus western blotting system
Manufactured by Thermo Fisher Scientific
Sourced in United States
The ECL Plus Western Blotting System is a laboratory equipment used for the detection and analysis of proteins in Western blot experiments. It utilizes an enhanced chemiluminescence (ECL) reaction to produce a luminescent signal that can be captured and quantified. The system provides a sensitive and reliable method for the visualization and quantification of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay reagent kit, by Thermo Fisher Scientific (6 mentions)
Cellytic extraction kit, by Roche (5 mentions)
Cyclin d1, by Cell Signaling Technology (3 mentions)
Anti rabbit linked to horseradish peroxidase, by Santa Cruz Biotechnology (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
P27kip1, by Cell Signaling Technology (2 mentions)
Gsk3β, by Cell Signaling Technology (2 mentions)
P gsk3β ser9, by Cell Signaling Technology (1 mentions)
Trek 1, by Santa Cruz Biotechnology (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
P21cip1, by Cell Signaling Technology (1 mentions)
Cyclin e1, by Cell Signaling Technology (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Bca protein determination kit, by Beyotime (1 mentions)
Image pro plus 6, by Media Cybernetics (1 mentions)
Nitrocellulose membrane, by Merck Group (1 mentions)
Horseradish peroxidase linked secondary antibody, by Abcam (1 mentions)
Bca protein assay kit, by Beyotime (1 mentions)
8 protocols using ecl plus western blotting system
1
Western Blot Protein Analysis
2
Western Blot Analysis of Transfected Cells
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The procedure was described previously [18 (link)]. In brief, after the transfection for 72 hrs, T24 cell and hFF cell protein was extracted using the CelLytic extraction kit supplemented with protease inhibitors (Roche, Basel, Switzerland). The total protein concentration was measured using the BCA Protein Assay reagent kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instruction. Equal amounts of cell lysate were electrophoresed on a 12% polyacrylamide gel, and the protein was transferred to an Immobilon-P PVDF membrane. Then, nonspecific binding was blocked at room temperature with 5% defatted milk. The membrane was then incubated with primary antibodies at 4°C overnight, followed by incubation at room temperature with a second antibody for 1 hr. Signals were detected by the ECL Plus Western Blotting System (Thermo Fisher Scientific, Waltham, MA).
3
Protein Extraction and Quantification
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The procedure was detailed previously 14 (link). In brief, cell protein samples were extracted and quantified using the CelLytic extraction kit supplemented with protease inhibitors (Roche, Basel, Switzerland) and the BCA Protein Assay reagent kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's instruction, respectively. Equal amounts of samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane. The membrane was incubated at 4℃overnight with primary antibodies, then followed by anti-rabbit horseradish peroxidase as second antibody. Signals were visualized using the ECL PlusWestern Blotting System (Thermo Fisher Scientific, Waltham, MA, USA). GAPDH was used as loading controls.
4
Western Blot Analysis of Signaling Proteins
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Total proteins were harvested using the CelLytic Extraction kit (Roche, Basel, Switzerland) containing protease inhibitors and then quantified using the BCA Protein Assay Reagent kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer's instructions. After separating proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis, protein was transferred to polyvinylidene fluoride membranes and then blocked in 5% fat-free milk. Using the primary antibodies and anti-rabbit linked to horseradish peroxidase (1∶5000) (Santa Cruz Biotechnology, Dallas, TX) as the secondary antibody, the target proteins were probed and then visualized using the ECL Plus Western Blotting System (Thermo Fisher Scientific, Waltham, MA). The blots were stripped and probed with α-β-actin antibody to confirm equivalent loading. The primary antibodies included the following: AKT (1∶1000), GSK3β (1∶500), cyclin D1 (1∶1000), PI3K (1∶1000), PTEN (1∶500) (Cell Signaling Technology, Boston, MA) and β-actin (1∶2000) (Santa Cruz Biotechnology, Dallas, TX).
5
Protein Extraction and Western Blot Analysis
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Total proteins were extracted using the CelLytic extraction kit containing protease inhibitors (Roche, Basel, Switzerland). Protein concentration was determined using the BCA Protein Assay reagent kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer's protocols. The proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transfer to polyvinylidene fluoride membranes. After blocking in 5% nonfat milk, the primary antibodies and anti-rabbit horseradish peroxidase second antibody (1:5000) were used to probe the target proteins. The bands were visualized using the ECL PlusWestern Blotting System (Thermo Fisher Scientific, Waltham, MA). GAPDH and β-tubulin were used as loading controls. The primary antibodies included the following: TREK-1 (1:1000), GAPDH (1:2000), β-tubulin (1:2000) (Santa Cruz Biotechnology, Dallas, TX), cyclin D1 (1:1000), cyclin E1 (1:500), CDK2 (1:500), p21 Cip1 (1:1000) and p27 Kip1 (1:1000) (Cell Signaling Technology, Boston, MA).
6
Western Blotting Analysis Protocol
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Western blotting analysis was performed as described previously.15 (link) Briefly, cells were harvested using the CelLytic extraction kit supplemented with protease inhibitors. Protein concentration was measured using the BCA Protein Assay reagent kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. After separated by SDS-PAGE in 12% (w/v) polyacrylamide gels, proteins were transferred to polyvinylidene fluoride membranes and blocked in 5% nonfat milk. Then, the primary antibodies and secondary antibodies were used to probe the target proteins. Immunoreactivity was then visualized by the ECL Plus Western Blotting System (Thermo Fisher Scientific).
7
Octreotide Modulates TLR4/NF-κB Signaling in CT26 Cells
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Mouse colon cancer CT26 cell line cells (Shanghai Guyan Industrial Co., Ltd., Shanghai, China; Lot No. 41596); Octreotide (Merck Biotechnology, Germany; Batch number: df-4854), mirVana miRNA isolation kit (Ambion Biotechnology, USA; Lot No. 63254.69), PrimeScript 1st strand cDNA Synthesis Kit (Dalian Baori Biotechnology, Dalian, China; Batch number: bj-63306); Power SYBR Green PCR Master Mix (Applied Biosystems Biotechnology, USA; Batch number: op-203.25); ABI 7500 (GM), Protease inhibitors (Sigma Biotechnology, Germany; Batch ot.: 632659.9); BCA protein determination kit (Beyotime Biotechnology, Shanghai, China; Batch number: as63695); TLR4, NF-κB p65 primary (1:500, 1:1,000; Thermo Fisher, USA; ab41901,69354), rabbit monoclonal glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1:1,000; Thermo Fisher, USA; Batch number: ab128015), horseradish peroxidase secondary antibody (1:2,000; Thermo Fisher, USA; Batch number: ab128015), ECL Plus Western Blotting System (Thermo Fisher, USA).
8
Western Blot Analysis of Protein Extracts
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Protein extracts were obtained by lysis buffer, and protein concentrations were measured using a BCA Protein Assay Kit (Beyotime Biotechnology, Haimen, P.R. China). Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to nitrocellulose membranes (Millipore). The membranes were blocked in 5% defatted milk for 1 h and then incubated with primary antibodies overnight at 4°C. Afterward, membranes were probed with horseradish peroxidase-linked secondary antibodies (1:2,000; Abcam) for 1 h. Protein bands were detected using an ECL Plus Western Blotting System (Thermo Fisher Scientific, Waltham, MA, USA). Band intensity was analyzed by Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA).
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