The total proteins were extracted using a protein Lysis buffer RIPA containing 1 mM PMSF (Solarbio; Beijing, China). The protein levels were normalized by probing the same blots with a β-actin antibody. The extracts were boiled with an SDS loading buffer and separated by SDS-PAGE. The membrane was incubated overnight at 4 °C with primary antibodies specific to anti-PDCD4 (CoWin Biosciences, Beijing, China), anti-β-actin (CoWin Biosciences, Beijing, China), anti-cleaved-CASP3 (Cell Signaling Technology Inc., Shanghai, China), anti-cleaved PARP (Cell Signaling Technology Inc., USA). Anti-immune rabbit IgG-HRP (Sungene Biotech, Tianjin, China) served as a secondary antibody. The intensity of each band was scanned and quantified using the image J software.
Anti β actin
Manufactured by CoWin Biotech
Sourced in China, United States
Anti-β-actin is a monoclonal antibody that specifically binds to the beta-actin protein, a widely expressed cytoskeletal protein found in eukaryotic cells. It is commonly used as a loading control or reference marker in various cell biology and biochemical techniques, such as Western blotting, immunocytochemistry, and flow cytometry.
Automatically generated - may contain errors
Lab products found in correlation
Bca kit, by Thermo Fisher Scientific (6 mentions)
Anti cleaved parp, by Cell Signaling Technology (2 mentions)
Ecl assay kit, by Beyotime (2 mentions)
Anti rabbit igg hrp, by Cell Signaling Technology (2 mentions)
Anti mouse igg hrp, by Cell Signaling Technology (2 mentions)
Anti cleaved casp3, by Cell Signaling Technology (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Solarbio (1 mentions)
Ab53707, by Abcam (1 mentions)
Enhanced chemiluminescence substrate, by Thermo Fisher Scientific (1 mentions)
Anti lamin a c, by Proteintech (1 mentions)
Anti stat3, by Cell Signaling Technology (1 mentions)
Anti ccl20, by Abcam (1 mentions)
Eecl western blot kit, by CoWin Biotech (1 mentions)
Bca protein assay kit, by CoWin Biotech (1 mentions)
Anti gfp, by Medical & Biological Laboratories (1 mentions)
Anti phospho p53 ser15, by Cell Signaling Technology (1 mentions)
Anti phospho chk2 thr68, by Cell Signaling Technology (1 mentions)
Anti survivin, by Abcam (1 mentions)
Anti caspase 9, by Abcam (1 mentions)
Anti cyclin a2, by Abcam (1 mentions)
9 protocols using anti β actin
1
Western Blot Analysis of Protein Expression
2
Immunoblotting of Cellular Proteins
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Whole proteins in HaCaT cells were extracted in radioimmunoprecipitation assay buffer (Runde Biologicals Ltd., Yichun, China) supplemented with 1 mM phenylmethylsulfonyl fluoride. Nuclear and cytoplasmic proteins were separated using a nuclear and cytoplasmic protein extraction kit (#P0027; Beyotime Biotechnology, Shanghai, China). Proteins were loaded onto 10% sodium dodecyl sulfate polyacrylamide gels and blotted onto polyvinylidene fluoride membranes, which were blocked with 5% bovine serum albumin for 1 h and then incubated with the following corresponding primary and horseradish peroxidase (HRP)-conjugated secondary antibodies: anti-K17 (1:1000; ab53707; Abcam, Cambridge, UK), anti-lamin A/C (1:1000; Proteintech, Phoenixville, PA, USA), anti-CCL20 (1:100; ab9828; Abcam), anti-STAT3 (1:1000; #124H6; Cell Signaling Technology) and anti-β-actin (1:5000; CoWin Biosciences, Cambridge, MA, USA). Specific bands were detected using an enhanced chemiluminescence substrate (Thermo Fisher Scientific, Waltham, MA, USA).
3
Quantification of Autophagy Levels in Yeast
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The YOM38 yeast cell containing the pR316-GFP-Atg8 plasmid yeast strain in glucose liquid medium with initial OD600 value of 0.1 was treated with 0, 0.3, 1, and 3 µM GENI or 300 µM RES for 22 h. The yeast cells of different groups were collected and sonicated for 5 min. Cell lysates were centrifuged at 12,000× g for 15 min to obtain the supernatants for Western blot. Protein concentrations were measured using a BCA protein assay kit (CoWin Biotech, Beijing, China). Western blot was performed as described in our previous study [20 (link)]. Approximately 20 µg protein was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto poly (vinylidene fluoride) membranes. Membranes were incubated with primary antibodies followed by secondary antibodies. The primary antibodies used were as follows: anti-GFP (Medical & Biological Laboratories, Nagoya, Japan) and anti-β-actin (CoWin Biotech, Beijing, China). The secondary antibodies used were horseradish peroxidase-linked antirabbit IgGs (CoWin Biotech, Beijing, China). Antigens were visualized using an eECL Western Blot Kit (CoWin Biotech, Beijing, China) and digitized using the ImageJ software (National Institute of Health, Rockville, MD, USA).
4
Immunoblotting Protein Expression Analysis
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Anti-phospho-AKT (Ser473), anti-AKT, anti-phospho-JAK2 (Tyr1007/Tyr1008), anti-JAK2, anti-phospho-Stat3 (Tyr705), anti-Stat3, anti-phospho-Chk2 (Thr68) and anti-phospho-p53 (Ser15) antibodies were purchased from Cell Signaling Technology (Boston, USA). Anti-Bax, anti-Bcl-2, anti-Mcl-1, anti-Caspase-3, anti-Caspase-9, anti-Survivin, anti-Cyclin A2, anti-phospho-ATM (Ser1981), anti-ATM, anti-γH2A.X (Ser139) and anti-LMP1 antibodies were purchased from Abcam (Cambridge, UK). Anti-pBad (S136) and anti-Bad were purchased from Bioworld Technology (Minneapolis, USA). Anti-p53 and anti-Zta antibodies were purchased from Santa Cruz Biotechnology (Texas, USA). Anti-β-actin and HRP-conjugated goat anti-mouse/rabbit secondary antibodies were purchased from ComWin Biotech (Beijing, China).
5
GUWE modulation of key signaling proteins
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The antibodies against IKKα, IKKβ, IkB, JNK, p38, ERK, NF-kBp65 and their phosphorylated antibodies were purchased from Cell Signaling Technology. Anti-β-actin and anti-histone were purchased from Beijing ComWin Biotech Co., Ltd (Beijing, China). Anti-mouse IgG-HRP and anti-rabbit IgG-HRP were obtained from Cell Signaling Technology.
DCs were treated with 40 μg/ml of GUWE for 0, 10, 30, 60 and 240 min and proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beijing ComWin Biotech Co., Ltd), then the protein concentration was determined by BCA Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Equal amount of proteins for each sample were transferred to PVDF membranes after isolation by 12% SDS-PAGE. After blocking with TBST buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% Tween 20) contained 5% nonfat milk for 1 h at RT, primary antibodies were added and incubated on shaker overnight at 4 °C, followed the incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. After extensive washing with TBST, the target proteins were detected using ECL assay kit (Beyotime Biotechnology Co., Ltd, China).
DCs were treated with 40 μg/ml of GUWE for 0, 10, 30, 60 and 240 min and proteins were extracted using Nuclear and Cytoplasmic Protein Extraction Kit (Beijing ComWin Biotech Co., Ltd), then the protein concentration was determined by BCA Kit (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. Equal amount of proteins for each sample were transferred to PVDF membranes after isolation by 12% SDS-PAGE. After blocking with TBST buffer (20 mmol/L Tris-HCl, 150 mmol/L NaCl, 0.05% Tween 20) contained 5% nonfat milk for 1 h at RT, primary antibodies were added and incubated on shaker overnight at 4 °C, followed the incubation with horseradish peroxidase-conjugated secondary antibodies for 1 h at RT. After extensive washing with TBST, the target proteins were detected using ECL assay kit (Beyotime Biotechnology Co., Ltd, China).
6
Liver Tissue Analysis of Nrf2 and CYPs
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Freshly isolated liver tissue was homogenized in lysis buffer supplemented with protease inhibitors (ComWin Biotech Co., Beijing, China). Nrf2 was extracted using a nuclear fractionation isolation kit (ComWin Biotech Co., Beijing, China). This homogenate was further mixed with buffer [60mM Tris-HCl, 2% sodium dodecyl sulfate (SDS) and 2% β- mercaptoethanol, pH 7.2] and boiled for 10 min. The protein content was determined using BCA protein assay reagent (Dingguo Changsheng Biotech Co., Beijing, China). A 50-μg sample of protein was applied to 10% SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane (Millipore, MA, USA) for 90 min. Membranes were blocked with 5% skimmed milk in Tris-buffered saline (TBST) and incubated with primary antibodies, including anti-CYP 1A2, anti-CYP 3A4, anti-GST A3, anti-Nrf2 (Biosynthesis Biotech Co., Beijing, China), and anti-β-actin (ComWin Biotech Co., Beijing, China), for 12 h at 4°C, followed by reaction with horseradish peroxidase-conjugated antibody for 1 h at 37°C. The detected bands were quantified using an image analyzer (ChemiScope 5600, Clinx Science Instruments, Shanghai, China), and β-actin was used as a loading control.
7
Apoptotic Pathway Analysis in H22 Cells
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Anti-caspase-3, anti-cleaved caspase-3, Anti-Bcl-2 and anti-Bax were purchased from Beyotime Biotech Co., Ltd. (Shanghai, China). Anti-caspase-7, anti-cleaved-caspase-7, anti-caspase-8, anti-cleaved-caspase-8, anti-caspase-9, anti-cleaved-caspase-9, anti-PARP, anti-cleaved PARP, anti-mouse IgG-HRP and anti-rabbit IgG-HRP were purchased from Cell Signaling Technology. Anti-β-actin was purchased from Beijing ComWin Biotech Co., Ltd. (Beijing, China).
H22 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) or 0.3% DMSO for 24 h. Cells were collected and lysed with the Cell Lysis Solution RIPA (Beijing ComWin Biotech Co., Ltd) for 30 min on ice. Samples were spun down (12,000 g for 15 min at 4 °C) to collect the supernatants and protein concentrations were measured by BCA Kit (Thermo Fisher Scientific, USA). Equal amount of protein in each sample was isolated by 12% SDS-PAGE and transferred to PVDF membranes (Biosharp, China). After blocking with TBST buffer contained 5% nonfat milk, membranes were incubated with corresponding primary antibodies and secondary antibodies conjugated to horseradish peroxidase (HRP), respectively. After washing with TBST, the target proteins were detected by ECL assay kit (Beyotime, China).
H22 cells were treated with different concentrations of CTPG (0, 100, 200, 300 and 400 μg/ml) or 0.3% DMSO for 24 h. Cells were collected and lysed with the Cell Lysis Solution RIPA (Beijing ComWin Biotech Co., Ltd) for 30 min on ice. Samples were spun down (12,000 g for 15 min at 4 °C) to collect the supernatants and protein concentrations were measured by BCA Kit (Thermo Fisher Scientific, USA). Equal amount of protein in each sample was isolated by 12% SDS-PAGE and transferred to PVDF membranes (Biosharp, China). After blocking with TBST buffer contained 5% nonfat milk, membranes were incubated with corresponding primary antibodies and secondary antibodies conjugated to horseradish peroxidase (HRP), respectively. After washing with TBST, the target proteins were detected by ECL assay kit (Beyotime, China).
8
Western Blot Analysis of Protein Extraction
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Total cell proteins were extracted using a Total Protein Extraction Kit (Applygen Technologies Inc., China) according to the manufacturer's instructions. Protein was quantified using the BCA Assay Kit (Beyotime, China). Protein samples were separated by electrophoresis on 10% SDS/PAGE gels and transferred to polyvinyl difluoride (PVDF) membranes (Thermo, USA). Antibodies were used as indicated by the manufacturer: anti-MRP4 (Abcam, USA), anti-β-actin (Beijing CoWin Biotech Co., Ltd., China).
9
Quantitative Western Blot Analysis of Cellular Proteins
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Protein concentration in cell lysate was determined using the Pierce bicinchoninic acid (BCA) assay kit (Thermo fisher, Rockford, IL, U.S.A.). The protein samples were boiled at 100°C for 5 min in 5× sample buffer (Beijing CoWin Biotech Co., Ltd., Beijing, China). The protein extracts (30 μg protein each) were electrophoresed in 6–15% SDS-polyacrylamide gels. The separated proteins were then transferred onto a nitrocellulose membrane (Pall Corp., Port Washington, NY, U.S.A.) in Tris–glycine buffer containing 20% methanol. The membranes were blocked and immunoblotted with a 1:1000 dilution of a primary antibody including anti-β-actin (Beijing CoWin Biotech Co., Ltd., Beijing, China, catalog number CW0096M), anti-S5a/PSMD4 (Cell Signaling Technology, Danvers, MA, U.S.A., catalog number 3846), and anti-CCK1R (Abcam, Shanghai, China, catalog number ab75153).
The proteins were detected using either goat anti-rabbit IgG (H+L)-HRP conjugated secondary antibody (1:3000) or goat anti-mouse IgG (H+L) secondary antibody (1:5000) with chemiluminescence (ECL) Western blot detection reagents (Bio-Rad, Hercules, CA, U.S.A.). β-Actin was used as the internal control. Western blots were developed and quantified using ImageJ software.
The proteins were detected using either goat anti-rabbit IgG (H+L)-HRP conjugated secondary antibody (1:3000) or goat anti-mouse IgG (H+L) secondary antibody (1:5000) with chemiluminescence (ECL) Western blot detection reagents (Bio-Rad, Hercules, CA, U.S.A.). β-Actin was used as the internal control. Western blots were developed and quantified using ImageJ software.
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