Cell lysates were subjected to SDS-PAGE and proteins transferred to PVDF membrane (Millipore) by electroblotting. The blots were blocked with 0.3% skim milk and 0.2% Tween 20 in PBS, and after incubating them with a primary antibody, they were washed with PBS containing 0.2% Tween 20 and incubated with a peroxidase-conjugated secondary antibody. Immunoreactive bands were detected by enhanced chemiluminescence or fluorescence with exposure to x-ray film or ODYSSEY® FC dual-imaging system (LI-COR Biotechnology) for quantifying the bands by using LI-COR Image Studio software.
Odyssey fc dual imaging system
Manufactured by LI COR
Sourced in Germany
The Odyssey FC Dual imaging system is a fluorescence imaging instrument designed for high-performance, two-color detection. It provides a sensitive and flexible platform for a wide range of applications, including western blotting, protein gels, nucleic acid gels, and cell-based assays.
Automatically generated - may contain errors
Lab products found in correlation
Pvdf membrane, by Merck Group (1 mentions)
Goat anti mouse irdye 680, by LI COR (1 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (1 mentions)
Irdye 800cw goat anti mouse, by LI COR (1 mentions)
Irdye 800cw goat anti rabbit, by LI COR (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
D7a2t, by Cell Signaling Technology (1 mentions)
Flag m2, by Merck Group (1 mentions)
Goat anti rabbit hrp, by Bio-Rad (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
β actin c4, by Santa Cruz Biotechnology (1 mentions)
Pngase f, by New England Biolabs (1 mentions)
3 protocols using odyssey fc dual imaging system
1
Western Blot Protocol for Protein Detection
2
Western Blot Analysis of Protein Expression
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After washing once with PBS, cells were resuspended in RIPA lysis buffer supplemented with cOmplete Protease Inhibitor (Roche) and incubated on ice for 30 min. Lysates were cleared by centrifugation at 21,000× g for 10 min at 4 °C. The protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions and 10–25 µg of protein were loaded on an SDS-PAGE gel. Gel electrophoresis was performed for 90 min at 110 V. Proteins were blotted onto a nitrocellulose membrane for 90 min at 450 mA. CD14 and GFP were detected with primary antibodies D7A2T and 4B10 (Cell Signaling Technology, 1:1000), respectively. The housekeeping protein β-actin was stained with primary antibody 926-42212 (LI-COR Biosciences, 1:3000). PIGH and PIGA were detected with a Flag-tag primary antibody (Genscript, A00187, 1:1000). As secondary antibodies, goat anti-rabbit IRDye800CW, goat anti-mouse IRDye800CW, and goat anti-mouse IRDye680 (LI-COR Biosciences, 1:10,000) were used. Blots were recorded on an Odyssey FC Dual imaging system (LI-COR Biosciences).
3
Immunoblot Detection of RNase Proteins
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Cells were resuspended in native lysis buffer supplemented with cOmplete Protease Inhibitor and incubated on ice for 30 min. Lysates were cleared by centrifugation (10,000 rcf, 5 min, 4 C). To deglycolysate proteins, cleared lysates and purified RNases were denatured and treated with PNGaseF (New England Biolabs, Frankfurt a.M., Germany) according to manufacturer's recommendations. RNaseT2 and RNase2 were detected with primary antibodies ab140191 (Abcam, Berlin, Germany; 1:1000)) and PA5-97305 (Thermo Fisher Scientific; 1:500), respectively. For standard immunoblot, secondary goat-anti-rabbit-HRP (Bio-Rad Laboratories, Feldkirchen, Germany; 1:5000) and Pierce ECL Subtrate (Thermo Scientific) were used, for quantitative immunoblot, secondary goat anti-rabbit IRDye800CW (Li-Cor Biosciences, Bad Homburg, Germany; 1:15,000) was used. Beta-actin (C4, Santa Cruz Biotechnology, Heidelberg, Germany; 1:1000), Flag (M2, Sigma-Aldrich; 1:1000) and HA (26183, Thermo Fisher Scientific; 1:1000) were detected with HRP-coupled primary antibodies and ECL substrate. Blots were recorded on an Odyssey FC Dual imaging system (Li-Cor Biosciences) .
Oligonucleotides RNA oligonucleotides were ordered from Biomers, Ulm, Germany and from Integrated DNA Technologies (IDT), Leuven, Belgium. DNA oligonucleotides were ordered from Integrated DNA Technologies (IDT), Leuven, Belgium.
Oligonucleotides RNA oligonucleotides were ordered from Biomers, Ulm, Germany and from Integrated DNA Technologies (IDT), Leuven, Belgium. DNA oligonucleotides were ordered from Integrated DNA Technologies (IDT), Leuven, Belgium.
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