Cells were lysed in RIPA buffer combined with proteasome inhibitor and phosphatase inhibitors (Beyotime). Equal amounts of proteins were separated by 10% or 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, then separated target proteins were transferred to a polyvinylidene fluoride membrane (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 60 min, the membranes were incubated overnight at 4 °C with antibodies specific to GAPDH (1:2000; Abcam, Shanghai, China), APELIN (1:2000; Abcam, Shanghai, China), RUNX2 (1:1600; Abcam, Shanghai, China), COL1A1 (1:1000; Abcam, Shanghai, China), non-phosphorylated (active) β-catenin (1:1000; Abcam, Shanghai, China), or total β-catenin (1:1000; Abcam, Shanghai, China). After washing with TBST three times (10 min each), the membranes were incubated with secondary antibodies (horseradish peroxidase-conjugated, Beyotime) for 1 h at room temperature. After washing three times with TBST, proteins were detected using enhanced chemiluminescence blotting reagents (Millipore). Signal intensity was measured by Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).
Enhanced chemiluminescence blotting reagents
Manufactured by Merck Group
Enhanced chemiluminescence blotting reagents are a specialized set of laboratory tools used in the detection and visualization of proteins in Western blot analysis. These reagents leverage the principle of chemiluminescence to enable sensitive and accurate protein detection and quantification.
Automatically generated - may contain errors
Lab products found in correlation
Xrs chemiluminescence detection system, by Bio-Rad (5 mentions)
Ripa buffer, by Beyotime (4 mentions)
Horseradish peroxidase conjugated secondary anti mouse or anti rabbit antibodies, by Beyotime (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Runx2, by Abcam (2 mentions)
Total β catenin, by Abcam (2 mentions)
Col1a1, by Abcam (2 mentions)
Proteasome and phosphatase inhibitors, by Beyotime (2 mentions)
Cst5174, by Cell Signaling Technology (2 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Apelin, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Ab223075, by Abcam (1 mentions)
Ab192256, by Abcam (1 mentions)
Ab246504, by Abcam (1 mentions)
Ab34710, by Abcam (1 mentions)
Ab128025, by Abcam (1 mentions)
Ab32572, by Abcam (1 mentions)
Protease and phosphatase inhibitor, by Beyotime (1 mentions)
Active β catenin, by Abcam (1 mentions)
5 protocols using enhanced chemiluminescence blotting reagents
1
Protein Expression Analysis by Western Blot
2
Western Blot Analysis of Cell Signaling Proteins
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Cells were lyzed in RIPA buffer supplemented with proteasome and phosphatase inhibitors (Beyotime). Equal amounts of proteins were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 1 h, the membranes were incubated overnight at 4°C with antibodies specific to GAPDH (1:1,500; CST#5174, Cell Signaling Technology, Danvers, MA, United States), COL1A1 (1:1,000; ab34710, Abcam, Cambridge, United Kingdom), RUNX2 (1:1,600; ab192256, Abcam), active β-catenin (1:1,000; ab246504, Abcam) or total β-catenin (1:1,000; ab223075, Abcam). A stripping method was used to measure the two antibodies of same molecular weight. After washing four times (5 min each time) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (Beyotime) for 1 h at room temperature. After washing three times (5 min each time) with TBST, proteins were detected using enhanced chemiluminescence blotting reagents (Millipore). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, United).
3
Western Blot Analysis of Cellular Proteins
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As previously reported (25 (link)). Cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitors (Beyotime). Equal amounts of proteins were separated by 10% SDS-PAGE and then transferred onto polyvinylidene flfluoride (PVDF) membranes (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 2 h, the membranes were incubated overnight at 4°C with antibodies specific to GAPDH (1:1,500; CST#5174, Cell Signaling Technology, Danvers, MA, United States), β-catenin (1:1,000; ab32572, Abcam), LC-3I/II(1:1,000; ab128025, Abcam). After washing four times (5 min each time) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (Beyotime) for 1 hr at room temperature. After washing three times (5 min each time) with TBST, proteins were detected using enhanced chemiluminescence blotting reagents (Millipore). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, United).
4
Western Blot Analysis of Osteogenic Markers
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Cells were lysed in RIPA buffer supplemented with proteasome and phosphatase inhibitors (Beyotime). Equal amounts of proteins were separated by 10% SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 1 hour, the membranes were incubated overnight at 4°C with antibodies specific to GAPDH (1:1,500; Cell Signaling Technology, Danvers, MA, USA), COL1A1 (1:1,000; Abcam, Cambridge, UK), RUNX2 (1:1,600; Abcam), active β-catenin (1:1,000; Abcam, Shanghai, China) or total β-catenin (1:1,000; Abcam). After washing four times (5 minutes each time) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with horseradish peroxidase-conjugated secondary anti-mouse or anti-rabbit antibodies (Beyotime) for 1 hour at room temperature. After washing three times (5 minutes each time) with TBST, proteins were detected using enhanced chemiluminescence blotting reagents (Millipore). Signal intensity was measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA, USA).
5
Western Blot Analysis of Osteogenic Markers
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Cells were lysed in RIPA buffer supplemented with proteasome and phosphatase inhibitors (Boster Biological Technology, Wuhan, China). Equal amounts of proteins were separated using 10% SDS-PAGE and transferred onto polyvinylidene uoride membranes (Millipore, Shanghai, China). After blocking in 5% non-fat milk for 1 h, the membranes were incubated overnight at 4°C with antibodies speci c against GAPDH (1:10,000), COL1A1 (1:1,000), RUNX2 (1:1,000), active β-catenin (1:1,000;), total β-catenin (1:1,000), phospho-GSK-3β (1:1,000), and total GSK3β (1:1,000). A stripping method was used to measure two antibodies of identical molecular weight. After washing four times (5 min each) in Tris-buffered saline with 0.1% Tween 20 (TBST), the membranes were incubated with a horseradish peroxidaseconjugated secondary anti-mouse or anti-rabbit antibody (Boster Biological Technology) for 1 h at room temperature. After washing three times (5 min each) with TBST, proteins were detected using enhanced chemiluminescence blotting reagents (Millipore). Signal intensities were measured using a Bio-Rad XRS chemiluminescence detection system (Bio-Rad, Hercules, CA).
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