RIPA lysis buffer containing protease inhibitor (Beyotime) was used to extract total protein samples from tissues and cells. The BCA kit (Beyotime) was used to quantify the protein concentration according to the manufacturer’s protocols. Then, the protein sample was separated with 10% SDS-PAGE and wet-transferred to PVDF membrane. After blocking with fat-free milk, the membrane was incubated with primary antibodies (anti-RHOB: ab53743, anti-GAPDH: ab8245; Abcam, Cambridge, MA, USA) and secondary antibody (ab97040; Abcam). Finally, the BeyoECL kit (Beyotime) was employed to visualize protein signals.
Beyoecl kit
Manufactured by Beyotime
Sourced in China, United States
The BeyoECL kit is a chemiluminescent detection system designed for Western blot analysis. It contains reagents necessary for the visualization of protein targets on a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Ab181602, by Abcam (10 mentions)
Ripa lysis buffer, by Beyotime (10 mentions)
Ab6721, by Abcam (9 mentions)
Bca kit, by Beyotime (6 mentions)
Anti gapdh, by Abcam (5 mentions)
Protease inhibitor, by Beyotime (5 mentions)
Pvdf membrane, by Merck Group (5 mentions)
Ab8245, by Abcam (3 mentions)
Bca protein assay kit, by Beyotime (3 mentions)
Ab40772, by Abcam (2 mentions)
Radioimmunoprecipitation assay lysis buffer, by Beyotime (2 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (2 mentions)
Anti sox2, by Abcam (2 mentions)
Ab125727, by Abcam (2 mentions)
Anti e cadherin, by Cell Signaling Technology (2 mentions)
Ab97040, by Abcam (1 mentions)
Ab17726, by Abcam (1 mentions)
Ab193555, by Abcam (1 mentions)
Ab16667, by Abcam (1 mentions)
Pvdf membrane, by Beyotime (1 mentions)
29 protocols using beyoecl kit
1
Western Blot Analysis of RHOB and GAPDH
2
Protein Expression Analysis by Western Blot
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Radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitor (Beyotime, Haimen, Jiangsu, China) was used as extract total proteins. After quantified using BCA kit (Beyotime), protein samples with equal amount were separated on 10% Sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) gel and then transferred to polyvinylidene fluoride (PVDF) membrane (Beyotime). The membranes were incubated with primary antibodies (rabbit anti-FOXP4: ab17726, rabbit anti-ki67: ab16667, rabbit anti-E-cadherin: ab40772, rabbit anti-Vimentin: ab193555, and rabbit anti- glyceraldehyde-3-phosphate dehydrogenase (GAPDH): ab181602; Abcam, Cambridge, Massachusetts) for overnight at 4°C after blocked with fat-free milk. Then, the membranes were incubated with horseradish peroxidase conjugated secondary antibody (ab6721, Abcam) at 37°C for 2 hours. The blot was visualized using BeyoECL kit (Beyotime) and analyzed with Image J version 1.42 software (glyceraldehyde-3-phosphate dehydrogenase (NIH), Bethesda, Maryland).
3
Western Blot Analysis of FZD8 Protein
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RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors (Beyotime) was employed to extract proteins from cultured cells. Equal amounts of protein lysates were separated using 10% SDS-PAGE and transferred to polyvi-nylidene fluoride membranes (Beyotime). Membranes were blocked with 5% skim milk, then incubated with primary antibodies (anti-FZD8: ab155093; anti-GAPDH: ab181602; both from Abcam, Cambridge, MA, USA) for overnight at 4°C. After washing with TBST, the membranes were incubated with secondary antibody (ab6721; Abcam) in room temperature at 1 h. Bands were visualized using BeyoECL kit (Beyotime) and ImageJ 1.42 software (NIH, Bethesda, MD, USA).
4
Protein Extraction and Western Blot Analysis
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Protein from cultured cells was extracted using radioimmunoprecipitation assay (RIPA)
lysis buffer (Beyotime, Haimen, Jiangsu, China). An equal amount of protein sample was
isolated using 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After
blocking with 5% fat-free milk, the membranes were incubated with primary antibodies
(anti-HOXB2: ab220390, anti-GAPDH: ab181602; Abcam, Cambridge, MA, USA) overnight at 4°C.
After incubation with horseradish peroxidase-conjugated secondary antibody (ab6721,
Abcam), the band signals were developed using BeyoECL kit (Beyotime). Expression of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control.
lysis buffer (Beyotime, Haimen, Jiangsu, China). An equal amount of protein sample was
isolated using 10% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After
blocking with 5% fat-free milk, the membranes were incubated with primary antibodies
(anti-HOXB2: ab220390, anti-GAPDH: ab181602; Abcam, Cambridge, MA, USA) overnight at 4°C.
After incubation with horseradish peroxidase-conjugated secondary antibody (ab6721,
Abcam), the band signals were developed using BeyoECL kit (Beyotime). Expression of
glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the internal control.
5
Western Blot Analysis of PAX2 in Cultured Cells
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Total protein from cultured cells was isolated using radioimmunoprecipitation assay lysis buffer (Beyotime, Haimen, Jiangsu, China) and protease inhibitors (Beyotime). Then, protein sample (50 μg) was isolated using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane. Membranes were incubated with primary antibody (1:1,000, rabbit monoclonal anti-PAX2: ab79389 [rabbit], 1:1,000, rabbit monoclonal anti-GAPDH: ab181602 [rabbit]; Abcam, Cambridge, Massachusetts) at 4°C for overnight after washing with (Tris-HCl buffer solution tween) TBST and blocked by 5% fat-free milk. Membrane was incubated with horseradish peroxidase–conjugated goat anti-rabbit secondary antibody (1:5,000, ab6721; Abcam) at room temperature for 2 hours. Protein signals were developed using BeyoECL kit (Beyotime). All the experiments were conducted in triplicates.
6
Western Blot Analysis of KCNE2 Protein
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Cultured cells were lysed with RIPA lysis buffer (Beyotime) according to the supplier's instructions to extract total proteins. Protein concentration was quantified with bicinchoninic acid Protein Assay kit (Beyotime). Equal amount of protein sample was separated using 10% sodium dodecylsulphate polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Beyotime). Membranes were incubated at 4°C for overnight with corresponding primary antibodies (anti‐KCNE2: ab69376; anti‐GAPDH: ab181602; Abcam, Cambridge, MA). Then, membranes were incubated with horseradish peroxidase‐conjugated secondary antibodies (ab6721, Abcam) at room temperature for 2 hr. Bands were visualized using BeyoECL kit (Beyotime) and analyzed with Image J 1.42 software (NIH, Bethesda, MD). Experiments were repeated in triplicates.
7
Western Blot Analysis of HMGB2 Protein
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Total proteins from cells were extracted using RIPA lysis buffer (Beyotime) and quantified with BCA kit (Beyotime). Twenty-five micrograms of protein sample was separated at 10% sodium dodecyl sulphate polyacrylamide gel electrophoresis and transferred to PVDF membrane. Then the membrane was incubated with primary antibodies (anti-HMGB2: ab124670, anti-GAPDH: ab181602, Abcam, Cambridge, MA, USA). PVDF membrane was further incubated with horseradish peroxidase-conjugated secondary antibodies (ab6721, Abcam). Band signal was detected with BeyoECL kit (Beyotime).
8
Protein Expression Analysis of Cultured Cells
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Protein sample of the cultured cells was isolated using radioimmunoprecipitation assay lysis buffer (Beyotime, Haimen, Jiangsu, China). After quantified with bicinchoninic acid protein concentration determination kit (Beyotime), equal amount of protein sample was isolated at 10% sodium lauryl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membrane. The membrane was blocked with fat-free milk, incubated with primary antibodies at 4°C for overnight and secondary antibody at 37°C for 4 hours. Band signals were developed using BeyoECL kit (Beyotime) and analyzed using the Image J version 1.42 software (National Institutes of Health, Bethesda, Maryland). The antibodies used in this work were anti-TRIM66: ab108445, anti-matrix metallopeptidase 9 (MMP-9): an73734, anti-glyceraldehyde 3-phosphate dehydrogenase: ab181602, and horseradish peroxidase conjugated goat antirabbit secondary antibody: ab6721; all were purchased from Abcam, Cambridge, Massachusetts.
9
Western Blot Analysis of Stem Cell Markers
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Proteins were resolved using 20% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by transfer of separated proteins onto a nitrocellulose membrane. Non-specific antigens on the membrane were blocked by incubating the membrane in 1×TBST (Tris-buffered saline with 0.1% Tween-20) containing 5% non-fat skim milk at room temperature for 1 h. Afterward, the membrane was incubated overnight with primary antibodies at 4°C followed by incubation with the secondary antibody (goat anti-mouse or anti-rabbit IgG antibody) at room temperature for 1 h. The primary antibodies used was anti-caspase3 (Abcam, US, 1;500), anti-Bax (R&D system, US, 1:1000), anti-Bcl2 (Abcam, US, 1;500), anti-Sox2 (Abcam, US, 1:1000), anti-Sox9 (Abcam, US, 1:1000), anti-CD133 (Proteintech, US, 1;1000), anti-Nanog (Proteintech, US, 1:1000), anti-SOCS2 (Abcam, US, 1:1000), anti-SOCS5 (Abcam, US, 1:500), anti-PTPN1 (Proteintech, US, 1:2000), anti-PTPN11 (Proteintech, US, 1:500), anti-STAT3 (Proteintech, US, 1:1000) The immunoblots were developed using the BeyoECL kit (Beyotime, China) and Tanon 5200 system (Tanon, China).
10
Western Blot Analysis of SYF2 Protein
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Proteins were isolated using RIPA lysis buffer (Beyotime, Haimen, China) and protease inhibitors (Beyotime). The concentration of extracted protein samples was analyzed with BCA kit (Beyotime), subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and then transferred to polyvinyl difluoride membranes (Beyotime). Membranes were blocked by 5% nonfat milk at room temperature for 4 hours and then incubated with primary antibodies (anti-SYF2: ab236417, anti-GAPDH: ab8245, both purchased from Abcam, Cambridge, Massachusetts) at 4°C for overnight, followed by incubation with goat anti-mouse secondary antibody (ab6789; Abcam) incubation at room temperature for 2 hour. Band signals were visualized using BeyoECL kit (Beyotime). GAPDH was used as internal control to normalize the expression of SYF2. Each sample was analyzed 3 times.
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