Following treatment with different concentrations (40, 50 and 60 µM) of 20(S)-PPD, protein from HepG2 cells was extracted by a radioimmunoprecipitation assay buffer containing 1% phenylmethane sulfonyl fluoride (both Beijing Dingguo Changsheng Biotechnology Co., Ltd.), then proteins were determined using a BCA protein assay kit. Proteins (20 µg/lane) were then loaded onto a 12% polyacrylamide-SDS gel. The gel was subsequently blotted onto a PVDF membrane and blocked with 5% (w/v) non-fat milk for 1 h at room temperature. The membrane was then incubated with cleaved caspase-3 (cat. no. 9664S), cleaved caspase-9 (cat. no. 7237T), PARP (cat. no. 9532T), Bcl-2 (cat. no. 4223T), Bax (cat. no. 5023T), Akt and p-Akt (Ser 473) were from the Phospho-Akt Pathway Antibody Sampler kit (cat. no. 9916T) and β-actin (cat. no. TA-09) primary antibodies at 1:1,000 dilutions at 4°C overnight. Primary antibody binding was detected using a secondary antibody conjugated to horseradish peroxidase The goat-anti-mouse (cat. no. IH-0031) and goat-anti-rabbit (cat. no. IH-0011) secondary antibodies were used at 1:5,000 dilution at room temperature for 1 h, and visualized using a BeyoECL Plus enhanced chemiluminescence kit (Beyotime Institute of Biotechnology) ImageJ software (version 1.5.0.26; National Institutes of Health, Bethesda, MD, USA) was used for analysis.
Beyoecl plus enhanced chemiluminescence kit
Manufactured by Beyotime
Sourced in China
The BeyoECL Plus enhanced chemiluminescence kit is a reagent used to detect and quantify proteins in western blot analysis. It generates a chemiluminescent signal proportional to the amount of target protein present in the sample.
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5 protocols using beyoecl plus enhanced chemiluminescence kit
1
Protein Extraction and Western Blot Analysis of 20(S)-PPD Treated HepG2 Cells
2
Protein Expression Analysis by Western Blot
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Protein extraction and Western blot were performed as described previously.7 Briefly, Western blot was performed for the detection of RXRα (cat. no. 3085S), E‐cadherin, N‐cadherin, β‐catenin, vimentin, Snail, Slug, claudin‐1 and ZEB‐1 (all from cat. no. 9782T), and GAPDH (cat. no. TA‐08). Cells were collected and lysed. Protein concentration was determined using the BCA protein assay kit. And proteins were loaded on polyacrylamide‐SDS gel (30 µg/lane), blotted onto a PVDF membrane and blocked with non‐fat milk for 1 hour at room temperature. The membrane was incubated overnight with primary antibody at 4°C, followed by incubation with HRP‐conjugated secondary antibody: goat‐anti‐mouse (cat. no. IH‐0031) and goat‐anti‐rabbit (cat. no. IH‐0011) at room temperature for 1 hour. The blots were visualized by BeyoECL Plus enhanced chemiluminescence kit (Beyotime Institute of Biotechnology; Jiangsu, China). ImageJ software was used for analysis.
3
Analyzing Epithelial-Mesenchymal Transition Markers
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Western blotting was performed to assess protein expression. Following treatment with PPD, the cells were harvested and lysed in radioimmunoprecipitation assay buffer (Beyotime Biotechnology) for 30 min on ice. The protein concentration was determined using a BCA protein assay kit according to the manufacturer’s protocol. Proteins (20 μL) were separated by 12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane, which was subsequently blocked with 5% (w/v) non-fat milk for 1 h at room temperature. Membranes were incubated with the appropriate primary antibodies against E-cadherin, vimentin, SIRT1, Slug, ZEB1, and GAPDH at a 1:1,000 dilution at 4°C overnight. Primary antibody binding was detected by incubation with a secondary antibody conjugated to horseradish peroxidase. The goat-anti-mouse and goat-anti-rabbit secondary antibodies (Beijing Dingguo Changsheng Biotechnology Co., Ltd.) were used at 1:5,000 dilution at room temperature for 1 h. The bands were visualized using a BeyoECL Plus enhanced chemiluminescence kit (Beyotime Institute of Biotechnology). ImageJ software (version 1.5.0.26; National Institutes of Health, Bethesda, MD, United States) was used for analysis.
4
Western Blot Assay for Protein Analysis
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The Western blot assay method is consistent with our previous report [41 (link)]. Cells or renal tissue were collected and lysed in cell lysis buffer (provided by Beyotime Biotechnology, Nantong, China) while maintaining a low temperature on ice. A BCA protein assay kit was utilized to measure protein levels. After separating protein samples (15 μL) using a 10% SDS-PAGE, they were transferred to a polyvinylidene fluoride membrane. Following this, the membrane was blocked with 5% (w/v) non-fat milk for 1 h at room temperature. The next step involved overnight incubation of the membranes at 4 °C with the correct primary antibodies. Detection of primary antibody binding was done by exposing the membranes to a secondary antibody linked to horseradish peroxidase for 1 h at room temperature. The bands were visualized by employing the BeyoECL Plus enhanced chemiluminescence kit manufactured by Beyotime Institute of Biotechnology (Nantong, China). The obtained data were analyzed using the ImageJ software (version 1.5.0.26; National Institutes of Health, Bethesda, MD, USA).
5
Molecular Mechanisms of Pseudo-G-Rh2 in A549 Cells
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Western blotting was performed to assess protein expression. Following treatment with pseudo-G-Rh2, A549 cells were harvested and lysed in radioimmunoprecipitation assay buffer (Beyotime Biotechnology, Jiangsu, China) for 30 min on ice. The protein concentration was determined using a BCA protein assay kit according to the manufacturer's protocol. Proteins (20 µl) were separated by 12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane, which was subsequently blocked with 5% (w/v) non-fat milk for 1 h at room temperature. Membranes were incubated with the appropriate primary antibodies against procaspase-3, procaspase-9, PARP, Bcl-2, Bax, Ras, p-Raf, Raf, p53, ERK, c-JNK, p38, p-ERK, p-JNK, p-p38 and β-actin at 1:1,000 dilution at 4°C overnight. Primary antibody binding was detected by incubation with a secondary antibody conjugated to horseradish peroxidase. The goat-anti-mouse (cat. no. IH-0031) and goat-anti-rabbit (cat. no. IH-0011) secondary antibodies (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) were used at 1:5,000 dilution at room temperature for 1 h. The bands were visualized using a BeyoECL Plus enhanced chemiluminescence kit (Beyotime Institute of Biotechnology). ImageJ software (version 1.5.0.26; National Institutes of Health, Bethesda, MD, USA) was used for analysis.
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