The protein lysate was analyzed by SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, Hercules, CA, USA). The membrane was blocked with 5% fat-free milk in PBST for 30 min, followed by incubation overnight at 4 °C with final dilution of primary antibodies against CXCL12 (Cat No. #3530), IL8 (Cat No. #94407), TGF-β (Cat No. #3711), HGF (Cat No. #52445), mTOR (Cat No. #2983), AKT (Cat No. #4685), AMPK (Cat No. #5831), LC3II/I (Cat No. #4108), ATG5 (Cat No. #12994), p-mTOR (Cat No. #5536), p-AMPK (Cat No. #50081), p-AKT (Cat No. #4060) or GAPDH (Cat No. #5174) all from CST (Beverly, MA, USA). Antibodies on membranes could be stripped using stripping buffer (Cat No. ab270550, Abcam, Cambridge, MA, USA) with gently shaking at 52 °C for 30 min for other blotting examination. Protein bands hybridized with primary and secondary antibodies on membranes were detected using ultrasensitive ECL chemiluminescence reagent (Beyotime Biotechnology, Shanghai, China) and exposed to film. Band intensity was quantified as the mean ± SD of three independent experiments.
Stripping buffer
Manufactured by Abcam
Sourced in United States
Stripping buffer is a laboratory reagent used to remove antibodies or other bound proteins from Western blot or immunoprecipitation membranes. It facilitates the reuse of the membrane for subsequent analyses.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Bio-Rad (2 mentions)
Ecl chemiluminescence reagent, by Beyotime (1 mentions)
Ecl plus immunoblotting detection reagents, by Thermo Fisher Scientific (1 mentions)
Hrp conjugated secondary antibody, by Proteintech (1 mentions)
Flag tag (flag), by ABclonal (1 mentions)
β catenin, by Proteintech (1 mentions)
Gapdh, by ABclonal (1 mentions)
Rabbit anti sox9 antibody, by Abcam (1 mentions)
Anti α tubulin, by Santa Cruz Biotechnology (1 mentions)
Ecl detection kit, by Thermo Fisher Scientific (1 mentions)
Anti β catenin antibody, by BD (1 mentions)
Anti β actin antibody, by Santa Cruz Biotechnology (1 mentions)
3 protocols using stripping buffer
1
Quantitative Western Blot Analysis
2
Protein Expression Analysis via SDS-PAGE
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The protein lysate was then analyzed by SDS-PAGE and transferred to PVDF membranes (Bio-Rad Laboratories, USA). The membrane was blocked with 5% fat-free milk in PBST for 30 min, followed by incubation overnight at 4°C with final dilutions of primary antibodies against β-catenin (Cat No. 17565-1-AP, Proteintech Group, USA), CAV1 (Cat No. A19006, Abclonal), GSK3β (Cat No. 12456, CST, Beverly, USA), p-GSK3β (Cat No. 5558, CST), VEGFB (Cat No. A2132, Abclonal), FGF1 (Cat No. A5900, Abclonal), PDGFB (Cat No. A1195, Abclonal), Flag (Cat No. AE005, Abclonal) or GAPDH (Cat No. A19056, Abclonal). Next, the membrane was washed three times and then incubated with HRP-conjugated secondary antibodies (Proteintech Group). Membranes could be stripped using stripping buffer (Cat No. ab270550, Abcam) at 52°C for 30 min via gently shaking and washed by PBST, then blocked by 5% fat-free milk for 2 h, and re-incubated by antibodies at 4°C overnight. The blotting bands were developed with ECL plus immunoblotting detection reagents (Thermo Fisher Scientific) using UVP Chemstudio Plus System (Analytik Jena, Germany), and captured using Image J.
3
Western Blot Analysis of Immortalized Cells
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After washing with PBS twice, immortalized cells were lysed using RIPA buffer (10 mM Tri-HCl, pH 7.4, 0.01% sodium dodecyl sulfate (SDS), and 0.1% Nonidet P-40 with protease inhibitors). The lysate protein concentration was measured by BCA protein assay and standardized by total protein using 3× sample buffer (Bio-Rad, Hercules, CA, USA). The samples were separated by SDS-10% polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Importantly, the protein on the membrane should be blocked with milk in PBS, treated with primary antibody, washed and incubated with horseradish peroxidase-conjugated secondary antibody. After following washes, the protein bands could be visualized by ECL detection kit (Thermofisher Scientific) under X-ray exposure. Afterwards, the membrane could be re-probed using internal reference primary antibody after incubation with stripping buffer (Abcam). The primary antibodies used were rabbit anti-SOX9 antibody (Abcam), anti-β-catenin antibody (BD Biosciences), anti-α-tubulin (Santa Cruz, Santa Cruz, CA, USA) and anti-β-actin antibody (Santa Cruz).
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