The MDA-MB-231 cells were seeded in 6-well plates at a concentration of 6.0 × 104 cells/well and treated with SchA (0, 40 and 80 µM) for 24 h. RIPA lysis kit (Beyotime) and protein analysis kit (Takara, Kyoto, Japan) were used for cell lysis and total protein quantification analysis, respectively. The total protein was then separated on 10% SDS-PAGE gel electrophoresis and afterward transferred onto a nitrocellulose membrane by electroblotting (Millipore, Billerica, MA, USA). After blocking the membrane using 5% BSA for 2 h at room temperature, it was incubated overnight at 4°C with primary antibodies (p-EGFR, PIK3R1, Cleaved-caspase 3, AKT1, p-AKT1 and MMP9), in a dilution of 1:1000, (Cell Signaling Technology, Danvers, MA, USA). They were then incubated with secondary antibodies at room temperature for 1 h. Later, the membrane was washed in three tris buffered saline + Tween (TBST) cycles for 10 minutes. The proteins were then visualized by adding ECL (Thermo) and subsequently scanned and imaged using a FluorChem FC 3 system (ProteinSimple, USA). Finally, image J software was used to analyze the results.
Electroblotting
Manufactured by Merck Group
Sourced in United States
Electroblotting is a laboratory technique used to transfer proteins or nucleic acids from a gel to a membrane, allowing for their detection and analysis. It serves as a core function in various analytical processes, enabling the immobilization of target molecules on a suitable support for further investigation.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Thermo Fisher Scientific (6 mentions)
Ripa buffer, by Beyotime (4 mentions)
Quantity one software, by Bio-Rad (3 mentions)
Protease and inhibitor mixes, by Thermo Fisher Scientific (2 mentions)
Hpa028543, by Merck Group (2 mentions)
Protein analysis kit, by Takara Bio (1 mentions)
P egfr, by Cell Signaling Technology (1 mentions)
Ripa lysis kit, by Beyotime (1 mentions)
Pik3r1, by Cell Signaling Technology (1 mentions)
Enhanced chemi luminescence (ecl), by Thermo Fisher Scientific (1 mentions)
Mmp 9, by Cell Signaling Technology (1 mentions)
P akt1, by Cell Signaling Technology (1 mentions)
Cleaved caspase 3, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride, by Merck Group (1 mentions)
Phospho stat3, by Santa Cruz Biotechnology (1 mentions)
Phospho c met, by Santa Cruz Biotechnology (1 mentions)
Stat3, by Santa Cruz Biotechnology (1 mentions)
Bcl 2, by Santa Cruz Biotechnology (1 mentions)
Cyclin d1, by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase conjugated secondary antibodies, by R&D Systems (1 mentions)
6 protocols using electroblotting
1
Schisandrin A Modulates EGFR/PI3K/AKT Pathway
2
Western Blot Analysis of HepG2 Cells
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HepG2 cells were lysed in RIPA buffer (Beyotime Institute of Biotechnology, Shanghai, China). Cell lysates (20 μg protein/lane) were loaded and separated on gradient polyacrylamide gels and then transferred to polyvinylidene difluoride membranes by electroblotting (Millipore Corp., Boston, MA, USA). Following blocking with 5% non-fat milk containing 0.3% Tween 20 for 1 h, the membranes were incubated overnight with primary antibodies at 4°C, including anti-hSulf-1 (1:250), -stat3 (1:500), -phospho-stat3 (1:500), -phospho-c-met (1:500), -bcl-2 (1:1000) and -cyclin D1 (1:500) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA). The membranes were washed three times with Tris-buffered saline containing Tween 20 and membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (R&D Systems China Co., Ltd., Shanghai, China) at 4°C for 1 h. Subsequently, membranes were exposed to enhanced chemiluminescent reagents for detection of protein bands. β-actin was used as an internal control.
3
Quantitative Western Blot Analysis
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Western blot was conducted according to previously described.17 RIPA buffer (Beyotime, China) containing protease inhibitor (Thermo Fisher Scientific, USA) was used to isolate total proteins. Then, the concentration of proteins was evaluated by BCA Protein Assay kit (Thermo Fisher Scientific). Sodium dodecylsulphonate (SDS) polyacrylamide gel electrophoresis was used to separate proteins, followed by electroblotting (Millipore, USA) which can transfer proteins onto a polyvinylidene fluoride (PVDF) membrane. Next, PVDF membranes were incubated with primary antibodies for a night at 4°C. The following antibodies were employed to examine target proteins: anti‐OTUB1 monoclonal antibody (1:500; ab175200, Abcam), anti‐EYA1 polyclonal antibody (1:500, ab194448, Abcam) and anti‐ubiquitin monoclonal antibody (1:500, sc8017, Santa Cruz). After incubation with secondary antibody (Proteintech, China) for 1 h at 37°C, protein band intensity was evaluated through Quantity One software (Bio‐Rad, USA).
4
Western Blot Analysis of OTUD3 and ACTN4
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Western blot was performed as previous study [15 (link)]. Extraction of total cellular proteins was extracted by RIPA buffer (Beyotime, Shanghai, China) containing protease and inhibitor mixes (Thermo Fisher Scientific, New York, USA) on ice. BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA) was performed to evaluate protein concentration. Equal amounts of proteins were separated by sodium dodecylsulfonate (SDS) polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride (PVDF) membrane by electroblotting (Millipore, Bedford, MA, USA). Primary antibodies were added and incubated throughout a night at 4° C. Primary antibodies including anti-OTUD3 monoclonal antibody (1:500; HPA028543, Sigma), anti-ACTN4 monoclonal antibody (1:1000, 15145, CST). After being incubated with the second antibody (CST, MA, USA) for 1h at room temperature, the intensity of protein bands was evaluated by Quantity One software (Bio-Rad, Hercules, CA, USA).
5
Protein Extraction and Western Blot Analysis
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Cells were collected and rinsed with PBS solution, and the protein was extracted by using the protein lysis buffer (including 1% NP-40, 1 μg/ml aprotinin, 1 μg/ml leupeptin, and 100 μg/ml PMSF). The protein concentration was assessed with a Bio-Rad DC Protein Assay (Bio-Rad, Hercules, USA). Then, 50 μg of proteins were used for sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Subsequently, the proteins were transferred to nitrocellulose membranes by electro blotting (Millipore Corp., Boston, USA). Membranes were blocked in 5% milk, washed with PBST (PBS with 0.1% Tween), and incubated with 1:1000 anti-tropomyosin antibody (cell signaling technology, Danvers, USA) at 4 °C for 12 h and 0.4 μg/ml anti-actin antibody (ZSBIO, Beijing, China) at 4 °C for 1 h. After the washing and incubation with 0.08 μg/ml horseradish peroxidase-conjugated anti-IgG antibody (ZSBIO, Beijing, China) at 4 °C for 1 h in 5% milk, the membranes were washed for three times and subjected to the enhanced chemiluminescent reagents (Millipore Corp., Boston, USA) for the distinction of protein bands.
6
Western Blot Analysis of Protein Targets
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Western blot was performed as previous study [12] . Extraction of total cellular proteins was extracted by RIPA buffer (Beyotime, Shanghai, China) containing protease and inhibitor mixes (Thermo Fisher Scientific, New York, USA) on ice. BCA Protein Assay kit (Thermo Scientific, Waltham, MA, USA) was performed to evaluate protein concentration. Equal amounts of proteins were separated by sodium dodecylsulfonate (SDS) polyacrylamide gel electrophoresis and transferred onto apolyvinylidene flusoride (PVDF) membrane by electroblotting (Millipore, Bedford, MA, USA). Primary antibodies were added and incubated throughout a night at 4°C. Primary antibodies including anti-OTUD3 monoclonal antibody (1:500; HPA028543, Sigma), anti-ACTN4 monoclonal antibody (1:1000, 15145, CST), anti-p65 monoclonal antibody (1:1000, 8242, CST), anti-p-p65 monoclonal antibody (Ser536)(1:1000, 3033, CST), anti-IκBα monoclonal antibody (1:1000, 4812, CST), anti-p-IκBα monoclonal antibody (Ser32) (1:1000, 2859, CST) and anti-Tubulin monoclonal antibody (code ab7291, 1:2000 dilution, Abcam). After being incubated with the second antibody (CST, MA, USA) for 1h at room temperature, the intensity of protein bands was analyzed by Quantity One software (Bio-Rad, Hercules, CA, USA).
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