Cells were lysed accordingly to protocols previously reported (3 (link), 5 (link)). Total protein concentration was assessed using Bio-Rad protein assay kit. The proteins of interest were determined separating equal amounts of proteins for each sample by SDS-PAGE and transferring to nitrocellulose membranes (Invitrogen, Carlsbad, CA). Incubation of membranes with the appropriate primary antibodies, was followed by incubation with IRDye-labeled secondary antibodies (680/800CW) (LI-COR Biosciences, NE), in order to visualize the immune complexes with the Odyssey infrared imaging system (LI-COR Corp., Lincoln, NE).
Irdye labeled secondary antibodies 680 800cw
Manufactured by LI COR
Sourced in Niger
IRDye-labeled secondary antibodies (680/800CW) are fluorescent-labeled antibodies used for the detection and visualization of target proteins in various biochemical and cellular assays. These antibodies are designed to bind to the primary antibodies that are specific to the target proteins, allowing for the detection and quantification of the target proteins using near-infrared (NIR) imaging technologies.
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4 protocols using irdye labeled secondary antibodies 680 800cw
1
Protein Extraction and Quantification
2
Immunoblotting Protocol for Protein Analysis
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Total cell lysates were obtained as previously described [44 (link), 45 (link)]. Total protein concentration was measured using the Bio-Rad Protein Assay kit, and cell lysates were separated by SDS-PAGE before transfer onto nitrocellulose membranes (Invitrogen, Carlsbad, CA). After immunoblotting with the appropriate antibodies, immune complexes were visualized by incubation with IRDye-labeled secondary antibodies (680/800CW) (LI-COR Biosciences, NE). For immunoblotting, the Odyssey infrared imaging system was used (LI-COR Corp., Lincoln, NE).
3
Quantifying Sclerostin, OxPhos, and MyHC II
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15 μg of protein from bone and muscle tissues were subjected to 12% SDS-PAGE and subsequently transferred to nitrocellulose membranes (Hybond, Amersham). The blots were probed using primary antibody anti-Sclerostin (Abcam), anti-OxPhos Complex IV subunit I (Invitrogen) and anti-MyHC II (Abcam) and IRDye-labeled secondary antibodies (680/800 CW) (LI-COR Biosciences). For immunodetection, the Odyssey infrared imaging system was utilized (LI-COR Corp., Lincoln, NE). All data were normalized to background and loading controls.
4
Quantitative Protein Analysis via Western Blot
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Protein amounts from all samples were assessed using the BCA-kit (Biorad) followed by protein concentration normalization before all western blot experiments. 30 μg of cell proteins was subjected to SDS-PAGE. Subsequently proteins were transferred to nitrocellulose membranes (Hybond, Amersham). The blots were probed using primary antibodies, described in Materials section, and IRDye-labeled secondary antibodies (680/800 CW) (LI-COR Biosciences). For immunodetection, the Odyssey infrared imaging system was utilized (LI-COR Corp., Lincoln, NE). All data were normalized to background and loading controls.
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