Cells were collected and then lysed in a RIPA lysis buffer with phenylmethanesulfonyl fluoride (PMSF) as previously described [31 (link)]. Protein concentration was detected with a BCA protein assay kit. The protein was separated on 8–12% SDS-PAGE Gels, and then transferred to a polyvinylidene fluoride (PVDF) membrane. After being blocked with 5% BSA at room temperature for 2 h, the PVDF membrane was incubated with a diluted primary antibody overnight at 4 °C. The next day, the PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibody (HRP-conjugated secondary antibodies) at room temperature for 2 h. Finally, the results were analyzed with the Super ECL prime (US Everbright®Inc., Suzhou, China) and western blotting detection system (Qin Xiang, Shanghai, China).
Super ecl prime
Manufactured by US Everbright
Sourced in China
Super ECL Prime is a chemiluminescent substrate designed for the detection of horseradish peroxidase (HRP) in Western blotting and other immunoassays. It provides a bright, stable signal with low background.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
Ripa lysis buffer, by Beyotime (3 mentions)
Cyclin d1, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Phenylmethylsulfonyl fluoride, by Thermo Fisher Scientific (1 mentions)
Propidium iodide, by Thermo Fisher Scientific (1 mentions)
M5655, by Merck Group (1 mentions)
Annexin 5 apc, by Thermo Fisher Scientific (1 mentions)
Wnt3a, by Cell Signaling Technology (1 mentions)
Gapdh, by Solarbio (1 mentions)
P ampkα, by Cell Signaling Technology (1 mentions)
Goat anti mouse igg, by Solarbio (1 mentions)
P p44 42 mapk, by Cell Signaling Technology (1 mentions)
P smad3, by Cell Signaling Technology (1 mentions)
Lc3a b, by Cell Signaling Technology (1 mentions)
Ripa lysis buffer, by Solarbio (1 mentions)
Goat anti rabbit igg, by Solarbio (1 mentions)
8 protocols using super ecl prime
1
Western Blot Analysis of Protein Expression
2
MTT Assay for Cell Viability
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3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT; M5655) and dimethyl sulfoxide (DMSO; D5879), were purchased from Sigma-Aldrich. RIPA lysis buffer, phenylmethylsulfonylfluoride (PMSF), and transfection reagent Lipofectamine 2000 were obtained from Thermo Fisher Scientific (New York, NY). Annexin V-APC (2005128) and propidium iodide (PI; 2048964) were obtained from Invitrogen. Super ECL prime (catalog no. S6008–100 mL) was purchased from US Everbright Inc. Anti-SP4 (sc-390124) antibody was purchased from Santa Cruz Biotechnology. CDK4 (#12790), CDK6 (#13331), Cyclin D1 (#2922), active Caspase-3 (#9661), and Wnt3a (#2721) were purchased from Cell Signaling Technology, Inc. The GAPDH (60004-1-Ig), β-catenin (51067-2-AP), PHF14 (ProteinTech, catalog no. 24787–1-AP, RRID: AB_2879724), and mouse IgG (ProteinTech, catalog no. B900620, RRID: AB_2883054) antibodies were purchased from ProteinTech Group.
3
Protein Extraction and Western Blot Analysis
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RIPA lysis buffer (Solarbio, Beijing, China) with Protein phosphatase inhibitor (Biomed, Beijing, China) and phenylmethylsulfonyl fluoride (PMSF, Biomed) was used to extract total protein lysate. Lysates were run on 10% SDS-PAGE gels, and protein bands were transferred to 0.45μm polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA), then blocked with 5% skim milk at room temperature for 1 hour. The membrane was incubated with primary antibodies (GNA15, Novus Biologicals, Centennial, USA, 1:1000; GAPDH, Solarbio, 1:1000; p-P38 MAPK, P38 MAPK, p-CREB, CREB, p-MAPKAPK2Thr222, MAPKAPK2Thr222, p53, cleaved-PARP, cleaved-Caspase3, p27 Kip1, Cyclin D1, CDK4, p-p44/42 MAPK, p-AMPKα, p-Akt, p-Smad3, LC3A/B, Cell Signaling Technology [CST], MA, USA, 1:1000) overnight at 4°C and probed with secondary antibodies (goat anti-rabbit IgG, goat anti-mouse IgG, Solarbio, 1:1000) at room temperature for 1 hour. The immunoreactive bands were defined using Super ECL Prime (US EVERBRIGHT, Suzhou, China).
4
AMPK-p53 Signaling Modulation in Cell Assays
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Cell culture reagents were purchased from GE and fetal bovine serum (FBS) was purchased from PAN-Biotech; DTT was purchased from Millipore (Massachusetts, USA); Compound C, AICAR, Resveratrol, Suramin, SNAP, and ODQ were purchased from Sigma-Aldrich; N6022, Pifithrin-α, VO-Ohpic trihydrate, AS1842856, and MK 2206 were from MCE. Hydrogen peroxide (H2O2) was from Sinopharm Chemical Reagent; 3-(4,5-dimethyl-2-thiazolyl)−2,5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Sangon Biotech; Lipofectamine® 3000 and Attractene were from Thermo Fisher Scientific and Qiagen, respectively. Fluor-de-Lys SIRT1 fluorometric drug discovery assay kit was from Enzo Life Sciences; Hydrogen Peroxide Assay Kit, Nitric Oxide Assay Kit, RIPA lysis buffer, Bicinchoninic Acid assay Kit, and Nuclear and Cytoplasmic Protein Extraction Kit were from Beyotime; Super ECL Prime was from US Everbright®Inc. The mRNA extraction kit was from Bioteke. Protein A Magnetic Beads and the Muse Count & Viability Assay Kit were purchased from Millipore. Anti-Flag M2 Magnetic Beads was from Sigma-Aldrich. Antibodies for phospho- and total-AMPK, acetyl- and total-p53, SIRT1, Cleaved- and total-Caspase-3, FOXO1, PTEN, β-actin, and acetylated-Lysine were from Cell Signaling Technology; antibodies for P21 was from Abcam; antibodies for Prdx2 was from Santa Cruz Biotechnology.
5
Western Blot Analysis of Protein Levels
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RIPA buffer (Applygen, C1053) containing 1% phenylmethanesulfonyl fluoride (PMSF) was used to harvest protein from the treated cells and protein levels were quantified using a BCA kit (Applygen, P1511). Equal amounts of protein (20–30 μg/lane) were then separated on 10% SDS-PAGE and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, IPVH00010 Immobilon-P Transfer Membrane) with a wet transfer system using 300 mA. Blots were blocked with 5% non-fat milk for 1 h at room temperature, followed by overnight incubation with primary antibodies (HO-1, Abcam, ab68477; GPX4, Abcam, ab125066) at 4°C. According to the antibody instructions, the concentration of the HO-1 and GPX4 antibodies diluted at the ratio of 1:10000 and 1:1,000 were 5 ng/ml and 420 ng/ml, respectively. After three washes with Tris-buffered saline containing Tween 20 (TBST), the membranes were incubated with HRP-conjugated secondary antibodies (ProteinTech, sa00001-2) for 1 h at room temperature. After three additional washes in TBST, Super ECL Prime (US EVERBRIGHT, S6008) was used to detect protein bands together with a Bio-Rad (United States) imaging system. ImageJ v1.8.0 (National Institutes of Health) was used for the densitometric analysis of the protein bands.
6
Immunoblotting Analysis of Signaling Proteins
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RIPA lysis buffer (Beyotime, Shanghai, China) supplemented with Protein phosphatase inhibitor (Biomed, Beijing, China) and phenylmethylsulfonylfluoride (PMSF, Biomed) was used for protein extraction. Lysates were run on 10% polyacrylamide gel electrophoresis (PAGE) gels, and protein bands were transferred to 0.45 μm polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA), then blocked with 5% skim milk at room temperature for 2 h. The membrane was incubated with primary antibodies (GAPDH, cleaved-PARP, cleaved caspase3, p27 Kip1, Cyclin D1, CDK4, p-AKT, AKT, PI3K, Cell Signaling Technology [CST], MA, USA, 1:1000; p-PI3K, Affinity Biosciences LTD, Jiangsu, China, 1:1000) overnight at 4 °C and probed with secondary antibodies (goat anti-rabbit IgG horseradish peroxidase (HRP), Zhongshan Golden Bridge Biotechnology, Beijing, China, 1:2000) at room temperature for 1 h. The immunoreactive bands were detected using Super ECL Prime (US EVERBRIGHT, Suzhou, China) according to the manufacturer’s protocol.
7
Protein Expression Analysis by Western Blotting
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Cells were lysed using ice-cold RIPA lysis buffer (Beyotime) and the protein concentrations were quantified. Western blotting assay was performed as previously described (32 (link)). The PVDF membranes were incubated with rabbit anti-cyclin D1 (1:1,000; Proteintech, Wuhan, China), c-Myc (1:1,000; Cell Signaling, Boston, MA, USA), cyclin A2 (1:1,000; Cell Signaling), β-catenin (1:1,000; Cell Signaling), MMP2 (1:1,000; Proteintech), MMP7 (1:1,000; Cell Signaling), BTRC (1:1,000; Cell Signaling), FBXW7 (1:1,000; Proteintech), and α-tubulin (1:2,000; Proteintech) primary antibodies at 4°C for overnight. After conjugated with HRP goat anti-rabbit IgG antibody (1:5,000; Abcam, Cambridge, MA, USA), immunoreactive bands were visualized by the Super ECL prime (US Everbright Inc., Suzhou, China) under a western blotting detection instrument (Clinx Science Instruments, Shanghai, China).
8
Western Blot Analysis of Epithelial-Mesenchymal Transition Markers
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Cell lysates were prepared by incubation for 5 min on ice with lysis buffer (RIPA buffer with 1% SDS and 1× protease inhibitor cocktail) and sonication for 3×8s pulses in a Soniprep 150 MSE, 30% power. Cell lysates were centrifuged at 4°C, 12000g for 15 min to remove debris. The protein concentration of the supernatants was determined by Pierce BCA Protein Assay Kit (Thermo Scienti c, #23227). Equal amounts of proteins were separated in SDS-PAGE by electrophoresis and transferred to a PVDF membrane (Millipore, #ISEQ00010). The membrane was blocked with 5% skim milk (Solarbio, #D8340) at room temperature and incubated with indicated antibodies overnight at 4°C. And then, the membrane was washed by TBST (1×TBS containing 0.05% Tween-20). The membrane was incubated with a corresponding secondary antibody according to the primary antibody at room temperature for 1h and washed as mentioned above. The blots were visualized using Super ECL Prime (US EVERBRIGHT ® INC, #S6008) by G-box (Syngene, Chemi XT). Intensity of Western blotting band was analyzed by ImageJ 1.46 software, and β-actin was used as a loading control to normalize the amount of protein. Primary antibodies used were antibodies against SNAIL (Cell Signaling Technology, #3879S), E-cadherin (BD Biosciences, #610181), β-actin (Abways Technology, #ab0035), and EIF4G1 (Proteintech, # 15704-1-AP).
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