Cell and tissue lysates were prepared and Western blot analysis was conducted as previously described (Rasmussen et al., 2008 (link)) using a Chemidoc XRS (Bio-Rad, Hercules, CA, USA) or a Li-Cor Odyssey CLx (Li-Cor biosciences, Lincoln, NE, USA), respectively. The following antibodies were used: GLUT1 (Cell Signaling Technologies, Danvers, MA, USA; Novus Biologicals, Littleton, CO, USA), GLUT2 (Abcam, Cambridge, MA, USA), GLUT4 (Cell Signaling Technologies), phospho-PDH (Abcam), and PDH (Abcam). Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) was used as a loading control for the data using a Chemidoc XRS. Total protein was stained using a REVERT total protein staining kit (Li-Cor biosciences) according to the manufacturer’s protocol to normalize the data when a Li-Cor Odyssey was used.
Revert total protein staining kit
Manufactured by LI COR
Sourced in United States
The REVERT total protein staining kit is a rapid and sensitive method for visualizing total protein on Western blots. The kit utilizes a fluorescent dye that binds to proteins, allowing for the detection and quantification of protein bands.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey clx, by LI COR (2 mentions)
Glut4, by Cell Signaling Technology (1 mentions)
Glut1, by Cell Signaling Technology (1 mentions)
Glut2, by Abcam (1 mentions)
Glyceraldehyde 3 phosphate dehydrogenase gapdh, by Santa Cruz Biotechnology (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Radioimmunoprecipitation assay buffer, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
β actin, by Merck Group (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Anti eea1, by Cell Signaling Technology (1 mentions)
Anti met, by Cell Signaling Technology (1 mentions)
Anti pten, by Cell Signaling Technology (1 mentions)
Tgx gel, by Bio-Rad (1 mentions)
Rnalater, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
P0044, by Merck Group (1 mentions)
Image studio software lite ver 5, by LI COR (1 mentions)
P8340, by Merck Group (1 mentions)
Irdye secondary antibody, by LI COR (1 mentions)
4 protocols using revert total protein staining kit
1
Western Blot Analysis of Glucose Transporters
2
Protein Expression and Quantification
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Whole-cell lysates were prepared with radio immunoprecipitation assay buffer (Cell Signaling Technology, Boston, MA, USA). Protein concentrations were determined using the Bradford protein assay (Bio-Rad, Hercules, CA, USA). Equal amounts of protein samples were loaded onto acrylamide gels and subjected to electrophoresis, followed by transfer to polyvinylidene difluoride (PVDF) membranes (Millipore, Burlington, MA, USA). Total protein staining was performed with a Revert Total Protein Staining Kit (LI-COR, Lincoln, NE, USA). PVDF membranes were then blocked with 5% nonfat milk and probed with antibodies recognizing His tag (1:2000, Novus Biologicals, Cat# NBP125939, Centennial, CO, USA), heme oxygenase-1 (HO-1, 1:1000, Enzo Life Sciences, Cat# ADI-SPA-896, Farmingdale, NY, USA), and β-Actin (1:3000, Sigma-Aldrich, Cat# A2228, St. Louis, MO, USA). After incubation in secondary antibodies (Cell Signaling) for 1 h, the membranes were incubated with ECL substrates (Pierce, ThermoFisher, Pittsburgh, PA, USA) and developed with X-ray film. ImageJ software (Version: 1.53k) was used for gel analyses.
3
Immunoblotting Analysis of Breast Cancer Cell Line
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MB-231 cells were collected in RNAlater (Life Technologies) and stored at −80°C. Frozen cells were placed in 2 mL collection tubes containing radioimmunoprecipitation assay buffer (1× PBS, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and 1× protease inhibitor). Cells were incubated for 10 min on ice and centrifuged for 10 min at 4°C (20,000 g), supernatant was collected, and 50 μg protein was resolved on 12% TGX gels (Bio-Rad) and transferred to polyvinylidene difluoride (PVDF) membranes. The Revert Total Protein Staining kit (Li-Cor) was used as the protein loading control, according to the manufacturer’s instructions. The following primary antibodies were used for detection: 2A8 anti-Ago (1:1,000, MABE56; Millipore), anti-MET (1:1,000, 4560; Cell Signaling), anti-PTEN (1:1,000, 9559; Cell Signaling), anti-EEA-1 (1:1,000, 2411; Cell Signaling), and anti-GAPDH (1:1,000, 2118; Cell Signaling). Protein detection was performed after incubation with secondary antibodies (Li-Cor), membranes were washed, and signal was acquired using Li-Cor Odyssey CLX (Li-Cor).
4
Protein Quantification and Western Blotting
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Total protein cell extracts were prepared in radioimmunoprecipitation assay buffer (RIPA buffer, 1% NP-40, 50 mM Tris pH 7.6, 120 mM NaCl, 1 mM EDTA) with protease and phosphatase inhibitors (Sigma-Aldrich P8340 and P0044), quantified using the Bicinchoninic acid protein assay method and analysed by SDS-PAGE as described previously [1 (link)]. Western blotting was performed with primary antibodies to PPARγ (C26H12), C/EBPα (2295), β−actin (8H10D10), active β-catenin (D13A1) (Cell Signalling Technologies ®, Leiden, The Netherlands), aP-2 (C-15), collagen type III alpha 1 (B-10 (Santa Cruz Biotechnology Inc) or collagen III (ab7778, Abcam PLC, Cambridge, UK) at 1:1000 dilution. IRDye® secondary antibodies (LI-COR™, Lincoln, NE, USA) for detection with an Odyssey FC image analyser (LI-COR®) was used and images were analysed with Image Studio Software Lite Ver 5.2 (LI-COR®). Quantitation was relative to nitrocellulose-bound total protein, stained using REVERT™ total protein staining kit (926-1101, LI-COR™) and analysed at 700 nm with the Odyssey FC image analyser, unless otherwise stated.
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