The U2OS and MG63 cells after different treatments were lysed in RIPA buffer supplemented with 1% protease inhibitors (Sigma). Protein concentrations were measured by BCA assay kit, and the protein samples were then denatured at 98°C for 10 min. Equal amounts of the denatured proteins were then subjected to electrophoresis on a 10% SDS-PAGE gel followed by transferring to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, USA). The PVDF membranes were incubated with 5% skimmed milk for 1 hrs at room temperature. Subsequently, the PVDF membranes were incubated with different primary antibodies against WNT2B (Abcam, Cambridge, USA), active β-catenin (Abcam), total β-catenin (Abcam), cyclin D1 (Abcam), c-myc (Abcam) and β-actin (Abcam) at 4°C overnight. After washing with Tris-buffered saline containing 0.1% Tween 20 for 3 times, the PVDF membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Abcam) for 2 hrs at room temperature. The protein bands were detected using an enhanced chemiluminescence kit (Thermo Fisher Scientific) and β-actin was used as the internal control.
Total β catenin
Manufactured by Abcam
Sourced in China
Total β-catenin is a protein that plays a central role in the Wnt signaling pathway. It functions as a transcriptional co-activator and is involved in the regulation of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Runx2, by Abcam (3 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Active β catenin, by Abcam (2 mentions)
Enhanced chemiluminescence blotting reagents, by Merck Group (2 mentions)
Xrs chemiluminescence detection system, by Bio-Rad (2 mentions)
Col1a1, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Wnt2b, by Abcam (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
β actin, by Abcam (1 mentions)
C myc, by Abcam (1 mentions)
Cyclin d1, by Abcam (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated, by Beyotime (1 mentions)
Apelin, by Abcam (1 mentions)
Gapdh, by Abcam (1 mentions)
Active β catenin, by Cell Signaling Technology (1 mentions)
5 protocols using total β catenin
1
Western Blot Analysis of Wnt/β-catenin Pathway
2
Protein Expression Analysis by Western Blot
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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).
3
Osteogenic Differentiation: Protein Analysis
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After 7 days of osteogenic induction, cells were washed with PBS and total protein was obtained by RIPA lysis buffer (Beyotime, China). Quantitative analysis was performed using BCA (Sigma, USA). Next, proteins were isolated on NuPAGE 10–12% polyacrylamide gel and transferred to a PVDF membrane (Millipore, USA). The membrane was blocked with 5% milk for 2 h and then incubated with primary antibodies overnight. The following primary antibodies were used: ALP (ABCAm, 1:400), RUNX2 (ABCAm, 1:400), total-β-catenin (ABCAm, 1:1000), active-β-catenin (Cell Signaling, 1:500), GSK-3β (ABCAm, 1:1000), p-GSK-3β (Santa Cruz, 1:500) and GAPDH (Cwbiotech, 1:500). The secondary antibody (Corning, 1:5000) was incubated according to the source of primary antibody, and the chemiluminescence ECL kit (Sigma, USA) was used for protein detection. ImageJ was used to analyze the corresponding spectral band intensity of scanned images.
4
Immunohistochemical Analysis of Cell Proliferation
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Paraffin-embedded serial sections were rehydrated, incubated in 5% H2O2 to block endogenous peroxidase activity and antigens were stained with hematoxylin and eosin (H&E), or subjected to immunohistochemical analysis for Ki67 antibody (tumor proliferation assay; Dako, Glostrup, Denmark) to evaluate the density of proliferating cells [58 (link)] and total β-catenin (Abcam). Ki67 and β-catenin primary antibodies were detected with biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) and peroxidase conjugated streptavidin (Dako), developed with 3,3′-diaminobenzidine (Vector Laboratories), counterstained with haemalaun, dehydrated and mounted in DPX (Merck, Darmstadt, Germany) and digitalized images were generated with a Nikon Eclipse 80i (Tokyo, Japan) microscope and analyzed using NIS Elements imaging software (Nikon). Results are expressed as relative percentage of Ki67–positive cells per field.
5
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).
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