Western blot analysis was performed as described earlier [47 (link)]. In brief, cells were lysed in a radio immunoprecipitation assay (RIPA) buffer. The protein concentration was determined by the standard Bradford assay. After blocking, the PVDF membranes were subsequently subjected to a Phosphate-Buffered Saline with Tween 20 0.05%, with a respective primary antibody at 4 °C overnight with constant rotation. Secondary species-specific antibodies were applied at the concentration of 1:10,000 for 1 h at room temperature. The signals were detected using the Pierce ECL Plus Western Blotting Substrate (Thermo Scientific, Waltham, MA, USA). Mild stripping buffer (Abcam, Cambridge, UK) was used to re-probe western blot membranes. Densitometry was carried out on three different experiments. The band intensity was measured using a ChemiDoc™ XRS+ gel documentation system (Bio-Rad, Hercules, CA, USA). Densitometry was performed with ImageJ (1.51 s) software (National Institute of Health, Bethesda, MD, USA) using β-tubulin as the loading control.
Mild stripping buffer
Manufactured by Abcam
Sourced in United Kingdom, United States
Mild stripping buffer is a solution used to remove antibodies from western blot membranes in a gentle manner, allowing for the reuse of the membrane for subsequent probing. This buffer is designed to dissociate antibody-antigen complexes without significantly disrupting the immobilized proteins on the membrane.
Automatically generated - may contain errors
Lab products found in correlation
Pierce ecl plus western blotting substrate, by Thermo Fisher Scientific (2 mentions)
Chemidoc xrs gel documentation system, by Bio-Rad (2 mentions)
Cell scraper, by Sarstedt (1 mentions)
Qproteome cell compartment kit, by Qiagen (1 mentions)
Okadaic acid, by Abcam (1 mentions)
Pierce enhanced chemiluminscent, by Thermo Fisher Scientific (1 mentions)
Bicinchoninic acid protein assay kit, by Beyotime (1 mentions)
Genetools software, by PerkinElmer (1 mentions)
P44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Cell extraction buffer, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico stable peroxide solution, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico luminal enhancer solution, by Thermo Fisher Scientific (1 mentions)
Protease phosphatase inhibitor cocktail, by Roche (1 mentions)
Np 40, by Merck Group (1 mentions)
5 protocols using mild stripping buffer
1
Western Blot Analysis of Cellular Proteins
2
Subcellular Fractionation and Western Blot Analysis
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MDA-MB468 and LNCaP cells were cultured for 72 h at normoxia or hypoxia. About 5x10^7 cells were washed with PBS (PAA) supplemented with protease inhibitor and harvested from culture flasks with a cell scraper (Sarstedt). After 10 min centrifugation at 500xg at 4°C, subcellular fractionation was conducted with the Qproteome Cell Compartment kit (Qiagen). Protein samples were precipitated in four volumes of ice-cold acetone and solved in PBS. Protein concentration was determined by bicinchoninic acid assays (BCA), using the BCA Protein Assay reagent (Pierce) with BSA standard dilutions for quantification. Samples were electrophoresed on 12% SDS polyacrylamide gels and blotted to a nitrocellulose membrane. Membranes were incubated in 5% non-fat dry milk for 1 h and washed in 0.1% PBS-T for 5 min. Primary antibodies were applied (Table 3 Table 3
3
Western Blot Analysis of Protein Phosphorylation
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Cells were lysed with NP-40 buffer (1% NP-40, 0.15 M NaCl, 50 mM Tris, pH 8.0) containing phosphatase inhibitors (okadaic acid; catalog no. ab120375; Abcam). Protein concentration was measured using a bicinchoninic acid protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). Equal amounts of protein (20 µg) were separated by sodium dodecyl sulfate-polyacryl-amide gel electrophoresis on 12% gels and transferred onto nitrocellulose membranes. After blocking with 5% non-fat milk in Tris-buffered saline containing Tween (TBST) for 1 h, membranes were incubated with primary antibody in TBST overnight at 4°C and subsequently incubated with anti-mouse (catalog no. A9309; Sigma-Aldrich; Merck Millipore, Darmstadt, Germany) or anti-rabbit (catalog no. ab97051; Abcam) horse radish peroxidase-conjugated secondary antibodies at a 1:2,000 dilution for 2 h at room temperature. Reactive proteins were detected using Pierce Enhanced Chemiluminscent and SuperSignal™ Chemiluminescent substrates (Thermo Fisher Scientific, Inc., Waltham, MA, USA). To control for loading efficiency, the blots were stripped with Mild Stripping Buffer (Abcam) at room temperature for 5–10 min and reprobed with GAPDH antibody (1:600).
4
Phospho-P44/42 MAPK Western Blot
5
Optimized Western Blot Protocol for Protein Analysis
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Western blot analysis was performed as described earlier [50 (link)]. In brief, cells were lysed in a modified radio immunoprecipitation assay (RIPA) buffer containing 50 mM Tris, pH 7.4, 1% NP-40 Sigma Chemicals, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF and protease/phosphatase inhibitor cocktail (Roche Molecular Biochemicals, Mannheim, Germany). The protein concentration was determined by the standard Bradford assay. The samples were run on 10% polyacrilamide gels and the proteins were transferred to PVDF membrane. After blocking the membranes were subsequently subjected to a Phosphate-Buffered Saline with Tween 20 0.05%, with a respective primary antibody (1:1000) at 4 °C overnight with constant rotation. Secondary species-specific antibodies were applied at the concentration of 1:10,000 for 1 h at room temperature. The signals were detected using the Pierce ECL Plus Western Blotting Substrate (Thermo Scientific). Mild stripping buffer (Abcam) was used to re-probe western blot membranes. Densitometry was carried out on three different experiments. The band intensity was measured using a ChemiDoc™ XRS+ gel documentation system (Bio-Rad, Hercules, CA, USA). Densitometry was performed with ImageJ (1.51 s) software (National Institute of Health, Bethesda, MD, USA) software using β-actin as the loading control.
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