Western blot was conducted to determine expression of Pgp in SKOV-3TR cell line and mouse model. The cells were lysed with 1X RIPA lysis buffer (Upstate Biotechnology, Charlottesville, VA) supplemented with complete protease inhibitor cocktail tablets (Roche Applied Science, IN, USA). Protein concentrations were evaluated by the DC Protein Assay (Bio-Rad, Hercules, USA). Equal amounts of proteins were separated by NuPAGE® 4–12% Bis-Tris Gel (Life Technologies), transferred onto nitrocellulose membrane (Bio-Rad), and incubated with specific primary antibodies (dilution: 1:1000) at 4°C overnight. After extensive rinsing with TBST, the membranes were further probed with respective secondary antibodies (LI-COR Biosciences, Lincoln, NE) at 1:20000 dilutions. Finally, the membranes were scanned using Odyssey® CLx equipment (LI-COR Bioscences) to detect bands. The density of the bands was then quantified by Odyssey software 3.0 (LI-COR Bioscences).
Respective secondary antibodies
Manufactured by LI COR
Sourced in United States
Respective secondary antibodies are laboratory reagents used to detect and amplify the signal from primary antibodies in various immunoassays and imaging techniques. They are designed to bind specifically to the constant region of primary antibodies, allowing for the visualization and quantification of target analytes.
Automatically generated - may contain errors
Lab products found in correlation
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (3 mentions)
Dc protein assay, by Bio-Rad (3 mentions)
Nitrocellulose membrane, by Bio-Rad (2 mentions)
Complete protease inhibitor cocktail tablet, by Roche (1 mentions)
Odyssey clx equipment, by LI COR (1 mentions)
Odyssey clx imaging equipment, by LI COR (1 mentions)
β actin, by Merck Group (1 mentions)
3 protocols using respective secondary antibodies
1
Pgp Expression in SKOV-3TR Cell Line and Mouse Model
2
Western Blot Analysis of ABC Transporters
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To evaluate the protein expression of Pgp and other ABC transporters including MRP1 and BCRP in different selected cell sublines, total proteins were extracted with 1 × RIPA lysis buffer (Upstate Biotechnology, Charlottesville, VA, USA). Protein concentrations were evaluated by the DC Protein Assay (Bio-Rad, Hercules, CA, USA), and western blot analysis was then performed as previously described (Duan et al, 2009b (link)). Briefly, 20 μg of total protein was resolved on NuPAGE 4–12% Bis-Tris Gel (Life Technologies) and transferred onto nitrocellulose membrane (Bio-Rad). The membrane was then blocked in Tris-buffered saline with 0.1% Tween-20 (TBST) containing 5% nonfat milk for 2 h and subsequently probed with primary antibodies (dilution: 1 : 1000) at 4 °C overnight. After washing three times with TBST, they were further incubated with respective secondary antibodies (LI-COR Biosciences, Lincoln, NE, USA) in a dilution of 1 : 20 000 for 1 h at room temperature. The membranes were washed again with TBST and rinsed with PBS. Finally, membranes were scanned using Odyssey CLx equipment (LI-COR Biosciences).
3
Quantitative Wee1 Protein Analysis
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