Cells and tumor tissues were lysed with protein extraction reagent (KeyGEN BioTECH, Jiangsu, China) according to the manufacturer’s instructions, and total cell lysate protein samples were obtained. Then samples were equally loaded on 10% SDS-polyacrylamide gel, electrophoresed, and transferred to polyvinylidene difluoride membranes (Bio-Rad). After blocking with 5% BSA in Tris-buffered saline (TBS) with 0.1% Tween 20 (TBST) for 2 hr, the membranes were incubated with the primary antibodies rabbit-polyclonal anti-human DUSP8 (1:2,000; Abcam, Cambridge, UK), rabbit-monoclonal anti-human ERK (1:1,000; Cell Signaling Technology (Danvers, MA, USA)), rabbit-polyclonal anti-human p-ERK (1:2,000; Cell Signaling Technology), rabbit-monoclonal anti-human AKT (1:1,000; Cell Signaling Technology), rabbit-monoclonal anti-human p-AKT (1:2,000; Cell Signaling Technology), or rabbit-monoclonal anti-human GAPDH (1:2,000; Cell Signaling Technology) at 4°C overnight. After the overnight incubation with the primary antibodies, membranes were washed in TBST three times and subsequently probed with a secondary anti-rabbit Ab-conjugated to horseradish peroxidase (HRP) for 1 hr (1:2,000; Cell Signaling Technology). Finally, the signals were detected and analyzed using the chemiluminescence image system (Bio-Rad).
Chemiluminescence image system
Manufactured by Bio-Rad
Sourced in United States
The Chemiluminescence image system is a laboratory equipment used to detect and analyze light-emitting chemical reactions. It captures and quantifies the luminescence signals produced by these reactions, providing a tool for researchers to study various biological and biochemical processes.
Automatically generated - may contain errors
Lab products found in correlation
Proteinase inhibitor, by Merck Group (2 mentions)
Rabbit polyclonal anti human p erk, by Cell Signaling Technology (1 mentions)
Rabbit monoclonal anti human akt, by Cell Signaling Technology (1 mentions)
Rabbit monoclonal anti human p akt, by Cell Signaling Technology (1 mentions)
Rabbit monoclonal anti human gapdh, by Cell Signaling Technology (1 mentions)
Protein extraction reagent, by Keygen Biotech (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Phos erk, by Cell Signaling Technology (1 mentions)
Goat anti rabbit igg h l hrp secondary antibody, by Abcam (1 mentions)
Phos akt, by Cell Signaling Technology (1 mentions)
Nf κb, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Gapdh, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
5 protocols using chemiluminescence image system
1
Western Blot Protein Expression Analysis
2
Protein Extraction and Western Blot
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Proteins were extracted from cells using RIPA with proteinase inhibitors (Sigma-Aldrich, USA) and the concentrations of proteins were measured using BCA Protein Assay kits (Thermo Scientific, MA). After separation by 10% SDS/PAGE, proteins were blocked with 3% non-fat milk for 1 h and immunoblotted with the primary antibodies at 4°C overnight. Proteins were incubated with secondary antibodies for 1 h. After TBST washing 3 times and the samples were measured using the chemiluminescence image system (Bio-Rad, USA).
3
Brain Tissue Protein Extraction and Western Blot
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After brain tissue was homogenates, total protein was extracted, and protein concentration was quantified using the BCA Protein Quantitation Kit. Total protein was mixed with 5 × SDS protein sample buffer solution (4:1) and heated at 100 °C for 10 min and stored at − 20 °C. Protein samples were subjected to SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad). The membranes were blocked with 5% skim milk in PBS plus 0.05% Tween 20 (PBS-T) at room temperature for 90 min, and then incubated with rabbit anti-mouse antibodies at appropriate dilution in TBST overnight at 4 °C. The primary antibodies used were as follows: RORα (1:1000; Abcam; no. ab60134), NF-κB (1:1000; Cell Signaling Technology; no. 4764), phos-NF-κB (1:1000; Cell Signaling Technology; no. 3039), ERK (1:1000; Cell Signaling Technology; no. 4695), phos-ERK (1:1000; Cell Signaling Technology; no. 4370), AKT (1:1000; Cell Signaling Technology; no. 4691), phos-AKT (1:1000; Cell Signaling Technology; no. 4060), and GAPDH (1:5000; Abcam; no. ab8245). PBS instead of primary antibody served as a control. After overnight incubation, the membrane was rinsed with PBS-T three times and incubated with Goat Anti-Rabbit IgG H&L (HRP) secondary antibody (1:2000; Abcam; no. ab6721) at room temperature for 1 h. Finally, the signals were determined by chemiluminescence image system (Bio-Rad).
4
Western Blot Analysis of LYC@FA-PEG-PLLA NPs
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Western blot was performed as described [29 (link)]. Briefly, after treatment with LYC@FA-PEG-PLLA NPs (0, 0.625, 1.25, 2.5 μM) for 24 h, cells were collected and lysed with RIPA lysis buffer (Radio immunoprecipitation assay buffer). The samples were quantified, and 5–10% SDS-PAGE gels were used to separate the protein. After this, the separated protein was transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA), and 5% skimmed milk was used to seal the membranes at room temperature for 1 h. After they were washed with PBS, the membranes were incubated overnight with the specific antibodies at 4 °C, followed by incubation for 1 h with the secondary antibody at room temperature. Finally, the results were observed through the Chemiluminescence image system (Bio-Rad, Hercules, CA, USA).
5
Protein Extraction and Western Blot Analysis
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Proteins were extracted from cells using RIPA with proteinase inhibitors (Sigma-Aldrich, USA) and the concentrations of proteins were measured using BCA Protein Assay kits (Thermo Scienti c, MA). After separation by 10% SDS/PAGE, proteins were blocked with 10% non-fat milk for 1 h and immunoblotted with the primary antiodies at 4 °C overnight. Proteins were incubated with secondary antibodies for 1 hr. After TBST washing 3 times and protein levels were measured using the chemiluminescence image system (Bio-Rad, USA).
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