The clinical tissues were homogenized with tissue homogenization treatment and centrifuged at 12000 g/min at 4°C for purification of protein. The cells were lysed with RIPA lysis buffer containing the protease inhibitor cocktail (Yeasen, Shanghai, China) on ice for 30 min. The lysis was centrifuged at 4°C for 15 min, and the supernatant was collected and subjected to BCA assay for protein concentration evaluation. A total of 30 μg protein was subjected to SDS-PAGE and transferred to the PVDF membrane. The membrane was incubated with primary antibody overnight and subjected to the second antibody at room temperature for 1 h. The antibodies are as follows: anti-RORγ (Abcam, Cambridge, USA, ab78007), anti-GAPDH (Proteintech, USA, 60004-1-Ig), anti-Myc (Santa Cruz, CA, USA, 9E10), anti-HBx (Abcam, Cambridge, USA, ab203540), and anti-β-actin (Proteintech, USA, 23660-1-AP). The enhanced chemiluminescence (ECL) system (Yeasen, Shanghai, China) was used to visualize the protein band. The Quantity One software (Bio-Rad, CA, USA) was used to quantify protein expression.
Ecl system
Manufactured by Yeasen
Sourced in China
The ECL system is a laboratory equipment used for chemiluminescent detection. It is designed to capture and analyze light emissions generated by chemical reactions, primarily for the purpose of protein and nucleic acid analysis.
Automatically generated - may contain errors
Lab products found in correlation
Amersham imager 600, by GE Healthcare (2 mentions)
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Amersham imager, by Cytiva (2 mentions)
Quantity one software, by Bio-Rad (1 mentions)
Protease inhibitor cocktail, by Yeasen (1 mentions)
Anti β actin, by Proteintech (1 mentions)
Anti myc, by Santa Cruz Biotechnology (1 mentions)
Anti hbx, by Abcam (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Ab65078, by Abcam (1 mentions)
Ab8227, by Abcam (1 mentions)
Horseradish peroxidase, by Jackson ImmunoResearch (1 mentions)
Nuclear and cytoplasmic protein extraction kit, by Keygen Biotech (1 mentions)
Goat anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Anti histone h3 antibody, by Abcam (1 mentions)
Anti cyclin d1, by Abcam (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Anti histone h3 acetyl k9 antibody, by Abcam (1 mentions)
5 protocols using ecl system
1
Protein Extraction and Analysis
2
Protein Extraction and Western Blot Analysis
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Whole cell lysates were extracted from cells after treatment by RIPA buffer (89901, ThermoFisher). For nuclear and cytoplasmic protein extraction, the proteins were extracted using the extraction buffers (KGP150, KeyGen Biotech). The concentration of protein was determined by BCA Protein Assay Kit (23227, ThermoFisher). Equal amounts of lysate protein were resolved on SDS-PAGE gels and transferred to nitrocellulose filter membrane followed by Western blot. Immunodetection was performed using an enhanced chemiluminescence ECL system (36222ES60, YEASEN). The binding affinity of LW-218 and drug targets was detected by Amersham Imager 600 (General Electric Company, Schenectady, NY, USA)24 (link). The blots are representative of multiple independent experiments.
3
Western Blot for SND1 Protein Detection
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Cell lysates were resuspended in radio-immunoprecipitation assay dissolution buffer. The whole-cell lysates (30–60 μg in each well) were separated using 10% dodecylsulfate, sodium salt (SDS)-polyacrylamide gel electrophoresis (SDS-PAGE) and subsequently transferred onto a polyvinylidene fluoride membrane (Millipore, Danvers, MA, USA). The membrane was blocked with 5% skimmed milk and incubated overnight with primary antibodies against SND1 (ab65078) or β-actin (ab8227) at 4°C (Abcam, Cambridge, MA, USA). This was followed by the addition of a secondary antibody conjugated with horseradish peroxidase (Jackson ImmunoResearch Laboratories, West Grove, PA, USA). The bands were observed by using an enhanced chemiluminescence (ECL) system (Yeasen, China).
4
Western Blot Protein Extraction and Analysis
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Whole proteins were extracted by RIPA buffer (89901, ThermoFisher,). Nuclear and Cytoplasminc proteins were extracted according to the instructions of Nuclear and Cytoplasmic Protein Extraction Kit (KGP150, KeyGEN, Nanjing, China). The concentration was determined by BCA Protein Assay Kit (23227, ThermoFisher). The proteins were separated on SDS-PAGE gels and transferred to NC membrane. After incubated with 3% BSA for 1 h, the target bands were incubated with the corresponding primary antibody and homologous secondary antibody. Finally, the bands were enhanced by ECL system (36222ES60, YEASEN, Shanghai, China) and visualized by Amersham Imager 600 (General Electric Company, Schenectady, New York, USA).
5
Nuclear and Cytoplasmic Protein Extraction and Analysis
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Nuclear and cytoplasmic protein extraction was performed using a nuclear and cytoplasmic protein extraction kit (Cat# MP1551-100T, MKBio, China), according to the manufacturer’s instructions. Protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, USA). Equal amounts of nuclear protein for each sample (30 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. Immunodetection was performed using an enhanced chemiluminescence ECL system (Cat# 36222ES60, Yeason, China). Histone H3 served as the loading control. The primary antibodies used in this study included anti-histone H3 antibody (1:1000, Cat# ab1791, Abcam, UK), anti-cyclin D1 (1:200, Cat# ab16663, Abcam, UK), HDAC3 antibody (1:1000, Cat# AF5349-50 μL, Affinity, China), anti-histone H3 (acetyl K9) antibody (1:500, Cat# ab32129, Abcam, UK), and FoxO3a (75D8) rabbit mAb (1:1000, Cat# 2497S, Cell Signaling Technology, USA). The secondary antibodies were goat anti-rabbit IgG HRP-linked antibody (1:2000, Cat# 7074S, Cell Signaling Technology, USA) and anti-mouse IgG HRP-linked antibody (1:2000, Cat# 7076S, Cell Signaling Technology, USA). The relative optical density of the protein bands was measured using the ImageJ software, and the protein levels were normalized to histone H3.
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