Lung cancer cells were lysed with RIPA buffer (Boston BioProducts, USA) supplemented with 1:100 Inhibitor Cocktail (ThermoFisher Scientific, USA) overnight at 4 °C. Protein concentration was measured using the Pierce Coomassie Protein Assay Kit (ThermoFisher Scientific, USA), and 30 μg of total protein was loaded into a 4–12% Bolt Bis-Tris Plus gel (ThermoFisher Scientific, USA), along with the protein marker Chameleon Duo ladder (LI-COR Biosciences, USA). Gels were electrophoresed at 110 mV and 4 °C for 75 min, and, using the iBlot 2 system (ThermoFisher Scientific, USA), proteins were transferred to a nitrocellulose membrane. The membrane was blocked with Odyssey blocking buffer (LI-COR Biosciences, USA) overnight at 4 °C and then incubated with the primary antibodies including mouse anti-human CD30 antibody (1:1500) and rabbit anti-human β-actin antibody (1:2000) for another 12 h at 4 °C. All primary antibodies were purchased from Novus Biologicals (Littleton, CO, USA). After washing with PBS-T (phosphate buffered saline with Tween 20), the membrane was incubated with secondary antibodies (donkey anti-mouse IRDye 800CW and goat anti-rabbit IRDye 680RD; LI-COR, USA) for 1 h at room temperature. The membrane was washed, scanned, and quantitatively analyzed using the Odyssey infrared Imaging System (LI-COR, USA).
Chameleon duo ladder
Manufactured by LI COR
Sourced in United States
The Chameleon Duo ladder is a molecular weight standard used in DNA gel electrophoresis. It provides a reference for determining the size of DNA fragments separated on an agarose gel.
Automatically generated - may contain errors
Lab products found in correlation
Bolt bis tris plus gel, by Thermo Fisher Scientific (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Pierce coomassie protein assay kit, by Thermo Fisher Scientific (2 mentions)
Odyssey infrared imaging system, by LI COR (2 mentions)
Inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw donkey anti mouse, by LI COR (1 mentions)
Iblot2 system, by Thermo Fisher Scientific (1 mentions)
Irdye 680rd goat anti rabbit, by LI COR (1 mentions)
Ripa buffer, by Boston BioProducts (1 mentions)
Ab9108, by Abcam (1 mentions)
Ab216773, by Abcam (1 mentions)
Immobilon fl membrane, by Merck Group (1 mentions)
Revert total protein stain, by LI COR (1 mentions)
Trupage lds sample buffer, by Merck Group (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
β actin antibody, by Novus Biologicals (1 mentions)
Iblot2, by Thermo Fisher Scientific (1 mentions)
3 protocols using chameleon duo ladder
1
Quantitative Analysis of CD30 Protein in Lung Cancer Cells
2
Quantitative Western Blotting Protocol
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Quantitative Western blotting was performed based on the method of Schütz et al. (65 (link)). Briefly, strains were grown for 16 h at 30 °C, 180 rpm shaking and 4 × 107 cells (equivalent to an OD600 of 4) were pelleted, washed in water, and resuspended in 1 mL 2 M lithium acetate. Cells were incubated on ice for 5 min, pelleted, resuspended in 200 µL 0.4 M NaOH, and incubated on ice for an additional 5 min. Cells were then centrifuged and resuspended in 100 µL TruPAGE LDS Sample Buffer (Sigma-Aldrich) containing 50 mM DTT and incubated at room temperature for 15 min.
Proteins were separated on a TruPAGE 10% polyacrylamide gel (Sigma-Aldrich) alongside the Chameleon Duo ladder (LI-COR) and transferred onto Immobilon-FL membrane (Merck Millipore). Total protein staining was performed using REVERT Total Protein Stain (LI-COR) following the manufacturer’s instructions.
The membrane was washed in PBS and blocked overnight in 0.1% Alkali-soluble Casein in 0.2× PBS. The membrane was then washed in PBS, incubated in Rabbit Anti-6X His tag antibody (ab9108; Abcam) (1:1,000) in blocking buffer +0.1% Tween20 for 1 h, washed four times in PBST, then incubated in Goat anti-Rabbit antibody conjugated to 800CW (ab216773; Abcam) (1:10,000) in blocking buffer +0.1% Tween20 for 3 h. The membrane was washed four times in PBST, then once in PBS before imaging on a LI-COR Odyssey Fc imaging system.
Proteins were separated on a TruPAGE 10% polyacrylamide gel (Sigma-Aldrich) alongside the Chameleon Duo ladder (LI-COR) and transferred onto Immobilon-FL membrane (Merck Millipore). Total protein staining was performed using REVERT Total Protein Stain (LI-COR) following the manufacturer’s instructions.
The membrane was washed in PBS and blocked overnight in 0.1% Alkali-soluble Casein in 0.2× PBS. The membrane was then washed in PBS, incubated in Rabbit Anti-6X His tag antibody (ab9108; Abcam) (1:1,000) in blocking buffer +0.1% Tween20 for 1 h, washed four times in PBST, then incubated in Goat anti-Rabbit antibody conjugated to 800CW (ab216773; Abcam) (1:10,000) in blocking buffer +0.1% Tween20 for 3 h. The membrane was washed four times in PBST, then once in PBS before imaging on a LI-COR Odyssey Fc imaging system.
3
Quantification of Pertuzumab Expression in Cancer Cells
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