RY10-4 (>95%) was prepared previously in our laboratory as described by Yuan et al. [5 (link)]. Sulforhodamine B (SRB) was purchased from J&K Scientific (Beijing, China). Dimethyl sulfoxide (DMSO) and N-acetylcysteine (NAC) were purchased from Sigma Aldrich (St. Louis, MO, USA). Hoechst 33342, propidium iodide (PI), reactive oxygen species assay kit (DCFH-DA), RIPA lysis buffer, BCA protein assay kit, and BeyoECL plus chemiluminescence kit were obtained from Beyotime Inc. (Shanghai, China). Annexin V-FITC/PI apoptosis detection kits was purchased from KeyGEN BioTECH (Nanjing, China). Specific antibodies against Bcl-2, Bax, p53, cyclin E, CDK2, cleaved caspase3, actin, STAT3, p-STAT3, p21, GAPDH and corresponding secondary antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA).
Beyoecl plus chemiluminescence kit
Manufactured by Beyotime
Sourced in China, United States
The BeyoECL Plus chemiluminescence kit is a laboratory equipment designed for the detection and quantification of protein expression levels through chemiluminescence assays. The kit provides the necessary reagents and components for performing these types of analyses.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
N acetylcysteine nac, by Merck Group (1 mentions)
P stat3, by Cell Signaling Technology (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Annexin 5 fitc pi apoptosis detection kit, by Keygen Biotech (1 mentions)
Cyclin e, by Cell Signaling Technology (1 mentions)
Gapdh, by Abcam (1 mentions)
Nf κb, by Abcam (1 mentions)
Snail, by Abcam (1 mentions)
Polyvinylidene fluoride (pvdf), by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Radioimmunoprecipitation lysis buffer, by Beyotime (1 mentions)
Electrochemiluminescent system, by PerkinElmer (1 mentions)
4 protocols using beyoecl plus chemiluminescence kit
1
Evaluation of RY10-4 Anti-cancer Effects
2
Western Blot Analysis of NF-κB, Snail, and GAPDH
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The treated or untreated cells were placed on ice and treated with RIPA lysate for 20 minutes. And the cells were collected in EP tubes and centrifuged to obtain protein samples. All protein samples were adjusted to the same concentration, and the polyacrylamide gel electrophoresis step was performed to separate the proteins with different molecular weights. Then, the protein on the gel was transferred to the nitrocellulose (NC) film. The hydrophobic binding sites on the nitrocellulose film were blocked with 5% BSA. After blocking, the membrane was added with primary antibody NF-κB (Abcam, 1: 1000), snail (Abcam, 1: 500), and GAPDH (Abcam, 1: 2000) and incubate at 4°C overnight. Then, added the corresponding horseradish peroxidase labeled secondary antibody to react in the dark for 1 hour. Finally, the protein was detected and photographed according to the operation of the BeyoECL Plus chemiluminescence kit (Beyotime).
3
Protein Extraction and Western Blot Analysis
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Cells and tissues were collected and processed for protein extraction. A total of 25 μg of each sample was resolved by 10% SDS-PAGE. Afterward, the proteins were transferred onto a 0.22 µm polyvinylidene difluoride membrane (PVDF, Millipore, USA). The antibodies used in immunoblotting are shown in Table S4 . A BeyoECL Plus chemiluminescence kit (P0018S, Beyotime) was applied to observe the protein bands. Grayscale analysis was performed with ImageJ (version 1.8.0). The experiments were repeated three times on different days.
4
Exosome Protein Profiling Using Western Blot
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A2780 cells and plasma-derived exosomes were lysed with radioimmunoprecipitation lysis buffer (Beyotime, Jiangsu, China), and total protein was obtained by centrifugation at 12,000×g for 15 min at 4 °C. After quantification using a bicinchoninic acid (BCA) Protein Assay Kit (Beyotime), equal amounts of cellular and exosomal protein were separated by SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membrane was blocked with 10% defatted milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h and then incubated overnight at 4 °C with primary antibodies, including anti-GM130 (Abcam ab52649; 1:1000), anti-HSP70 (Abcam ab2787; 1:1000), and anti-CD9 (Abcam ab236630; 1:1000). After washing with TBST three times, the membrane was incubated with appropriate HRP-conjugated secondary antibodies: goat anti-rabbit IgG H&L (HRP) (Abcam ab97051; 1:2000) or goat anti-mouse IgG H&L (HRP) (Abcam ab205719; 1:2000), as appropriate, for 1 h at room temperature. Finally, the protein bands were visualized on photographic films using the BeyoECL Plus chemiluminescence kit (Beyotime) with an electro-chemiluminescent system (PerkinElmer Life Science, Waltham, MA, USA).
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