Total protein in tissues and cells was extracted and protein concentration was determined via bicinchoninic acid kits (BOSTER Biological Technology Co., Ltd., Hubei, China). The extracted proteins were conducted with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (BOSTER Biological Technology) and transferred onto polyvinylidene fluoride membranes, which were blocked with 5% bovine serum albumin for 1 h. Then, the membranes were incubated with primary antibodies DACT2 (1:1000), active-β-catenin (1:5000), Glut1 (1:1000), β-actin (1:1000, all from Abcam Inc., MA, USA), p-β-catenin (1:1000), and lactate dehydrogenase A (LDH-A, 1:1000, both from Cell Signaling Technology, MA, USA) at 4 °C overnight. Next, the membranes were incubated with relative secondary antibody (Shanghai Miaotong Biotech Co., Ltd., Shanghai, China) for 1 h and developed using enhanced chemiluminescent reagent and the Bio-Rad Gel Doc EZ imager (Bio-Rad Laboratories, Hercules, CA, USA). Image J software (National Institutes of Health, Bethesda, Maryland, USA) was employed to analyze the gray values of the protein bands.
Active β catenin
Manufactured by Abcam
Sourced in United States
Active β-catenin is a protein that plays a key role in the Wnt signaling pathway, which is involved in various cellular processes such as cell proliferation, differentiation, and survival. This product provides a tool to detect and quantify the active form of β-catenin.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (3 mentions)
β actin, by Abcam (2 mentions)
Cyclin d1, by Abcam (2 mentions)
C myc, by Abcam (2 mentions)
Total β catenin, by Abcam (2 mentions)
Ripa buffer, by Beyotime (2 mentions)
Bicinchoninic acid kit, by Boster Bio (1 mentions)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis, by Boster Bio (1 mentions)
Glut1, by Abcam (1 mentions)
Gel doc ez imager, by Bio-Rad (1 mentions)
Lactate dehydrogenase a ldha, by Cell Signaling Technology (1 mentions)
P β catenin, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Abcam (1 mentions)
Enhanced chemiluminescence kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
β actin, by Thermo Fisher Scientific (1 mentions)
Wnt2b, by Abcam (1 mentions)
Hrp conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Abcam (1 mentions)
Nanog, by Abcam (1 mentions)
5 protocols using active β catenin
1
Quantitative Protein Analysis in Cells
2
Western Blot Analysis of Wnt/β-catenin Pathway
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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.
3
Western Blot Analysis of Stem Cell Markers
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Total protein was extracted from cells using cell lysis buffer. Proteins were loaded and separated on a 10% SDS-PAGE gel and then transferred to PVDF membranes. After blocking in 50 g/L non-fat milk in TBST (20 mmol/L Tris-HCl, 137 mmol/L NaCl, 1 g/L Tween 20, pH 7.6) for 2 h at room temperature, the membranes were incubated at 4°C overnight with the following primary antibodies: PTEN, AKT, p-AKT, β-catenin, active β-catenin, Nanog and GAPDH (Abcam). The membranes were then incubated for 1 h with HRP-conjugated secondary antibodies (Invitrogen, Logan, UT, USA). Finally, the membranes were visualized using the ECL-Plus detection system (Bio-Rad, Hercules, CA, USA).
4
Western Blot Analysis of Protein Expression
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Protein was harvested from the cells after transfection and lysed with RIPA buffer containing protease inhibitor (Beyotime, China). The concentration of proteins was measured using a BCA Protein Assay Kit (Pierce, USA). Equal amounts of proteins (40 μg) were separated with 10% SDS-PAGE (Pierce, Rockford, IL) and electrotransferred onto a PVDF membrane (Millipore). After being blocked with 5% non-fat milk at room temperature for 1 h, the membranes was treated with the following primary antibodies: Total β-catenin (1: 500, CST, USA), active-β-catenin (1: 500, Abcam, Cambridge, UK), cyclinD1 (1: 2000, Abcam), c-Myc (1: 1000, Abcam), p21 (1: 1000, Abcam), E-cadherin (1: 1000, Abcam), Vimentin (1: 1000, Abcam), and N-Cadherin (1: 1000, Abcam) overnight at 4°C. After being washed with PBST, the membranes were incubated with secondary antibodies at 37°C for 1 h. Protein blots were quantified using chemiluminescence method (Pierce, Rockford, IL) in accordance with the user’s manual.
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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