Cells were lysed in RIPA buffer containing 20 mM Tris (pH 8.0), 137 mM NaCl, 10% glycerol, 1% NP-40, 0.1% SDS, 0.5% deoxycholate, and 0.2 mM PMSF and 1X general protease inhibitor. The protein concentrations of whole-cell lysates were determined using the Thermo Fisher BCA assay. 6× sample buffer (300 mM Tris-HCl, pH 6.8, 60% glycerol, 30 mM dithiothreitol, 6% sodium dodecyl sulfate (SDS)) was added to yield a final concentration of 1× and lysates were boiled at 95° C for 10 min. Samples were subjected to SDS/PAGE and transferred to Immobilon PVDF membranes (Millipore) using a transblot turbo system (Biorad). Membranes were blocked in 5% BSA diluted in Tris-buffered saline. The following primary antibodies were incubated overnight at 4° C: anti-Chk1 (R&D), anti-Chk2 (Novus), anti-Wnt3a (R&D), and anti-β-actin (Abcam, loading control). Blots were washed and then incubated with secondary antibodies for 1 h at RT. Blots were washed and visualized by chemiluminescence using Clarity Western ECL (Biorad).
Anti wnt3a
Manufactured by R&D Systems
Anti-Wnt3a is a recombinant antibody that specifically binds and neutralizes the Wnt3a protein. Wnt3a is a secreted signaling protein that plays a role in various cellular processes, including cell fate determination, cell migration, and embryonic development. The Anti-Wnt3a antibody can be used in research applications to study the function and regulation of the Wnt3a signaling pathway.
Automatically generated - may contain errors
Lab products found in correlation
Anti β catenin, by Cell Signaling Technology (2 mentions)
Anti gsk3β, by Cell Signaling Technology (2 mentions)
Anti gapdh antibody, by GeneTex (2 mentions)
Anti wnt8a, by R&D Systems (2 mentions)
Hrp conjugated antibody, by Santa Cruz Biotechnology (2 mentions)
Dc protein assay, by Bio-Rad (2 mentions)
Luminol reagent, by Santa Cruz Biotechnology (2 mentions)
Immobilon pvdf membrane, by Merck Group (1 mentions)
Clarity western ecl, by Bio-Rad (1 mentions)
Trans blot turbo system, by Bio-Rad (1 mentions)
Anti β actin, by Abcam (1 mentions)
Anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti mouse igg, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride (pvdf), by Bio-Rad (1 mentions)
4 protocols using anti wnt3a
1
Cell Lysis and Western Blot Analysis
2
Western Blot Analysis of Wnt Pathway Proteins
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Cells were lysed in RIPA buffer containing protease and phosphatase inhibitors. The cell suspension was centrifuged at 15,000g for 15 min to remove debris, and protein levels in the supernatant quantified with BioRad’s DC protein assay. Proteins (5–10 μg) were electrophoresed and transferred per standard methods, prior to blocking in 5% milk/TBST. All primary antibodies were diluted 1:1000 in TBST and were incubated overnight at 4 °C. The antibodies included anti-β-catenin (Cell Signaling no. 9562), anti-GSK3β (Cell Signaling no. 9315), anti-Wnt3a (R&D Systems no. MAB1324-050), and anti-Wnt8a (R&D Systems no. AF2248). Anti-GAPDH antibody (Genetex no. GT-239) was used as a loading control. The appropriate secondary HRP-conjugated antibody (1:2000 dilution, Santa Cruz) was incubated with the blot and proteins were detected using Luminol reagent (Santa Cruz). Band intensity was determined by densitometry.
3
Western Blot Analysis of Wnt Pathway
4
Western Blot Detection of Wnt3a
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Samples were resolved by SDS-PAGE on a 12% gel and were transferred onto polyvinylidene difluoride membranes (PVDF; Bio-rad). Membranes were incubated with primary antibodies (anti-Wnt3a, R&D Systems) overnight at 4°C and detection was accomplished with secondary antibodies (e.g., anti-rat, IgG Molecular Probes; anti-rabbit IgG, Molecular Probes; anti-mouse IgG, Cell Signaling) conjugated to alkaline phosphatase or horseradish peroxidase. Protein bands were developed with 5-bromo-4-choloro-3-indoyl phosphate (BCIP; Sigma) and nitro blue tetrazolium (NBT; Sigma).
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