Ovaries from mice at day10 of age were treated with 10 μM MHY1485 (Millipore, Bedford, MA) for 3h and proteins were extracted using M-PER Mammalian Protein Extraction Reagent (Thermo, Rockford, IL) containing a protease inhibitor cocktail (Thermo). Protein concentrations in supernatants were determined by the bicinchoninic acid method (Pierce, Rockford, IL, USA). Equal amounts of protein lysates were loaded on 4–12% NuPAGE Bis-Tris gels (Invitrogen, Carlsbad, CA) in MOPS buffer and transferred to 0.45 μM pore nitrocellulose membranes (LI-COR, Lincoln, NE, USA). First antibodies were from Cell Signaling (Beverly, MA; #5536 for phosphor-mTOR; #2983 for mTOR; #9205 for phosphor-S6K1, #2708 for S6K1; #4858 for phospho-rpS6 (S235/6) and #2217 for rpS6) and IRdye 800 rabbit secondary antibody (926–32211) was from LI-COR. Images were generated using a LI-COR Odyssey infrared imager.
Irdye 800 rabbit secondary antibody
Manufactured by LI COR
Sourced in United States
The IRDye 800 rabbit secondary antibody is a fluorescent-labeled detection reagent designed for immunoblotting and immunohistochemistry applications. It is a highly specific antibody that binds to rabbit primary antibodies, allowing for visualization and detection of target proteins.
Automatically generated - may contain errors
Lab products found in correlation
Odyssey infrared imager, by LI COR (2 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nupage bis tris gel, by Thermo Fisher Scientific (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Mhy1485, by Merck Group (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Image studio lite, by LI COR (1 mentions)
2 protocols using irdye 800 rabbit secondary antibody
1
Ovarian mTOR Signaling Regulation
2
Quantification of mTOR Signaling Proteins
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In vitro-cultured ovaries were collected after a 1-h exposure to Compound C, followed by protein extraction using the Pierce BCA protein assay kit (Thermo Scientific) containing a protease-inhibitor cocktail and concentration determination. Protein samples were loaded onto a 4-12% NuPAGE Bis-Tris gel (Invitrogen), and then transferred to polyvinylidene fluoride membranes. The membranes were subsequently incubated with primary antibodies for TSC2, mTOR, S6 and eIF4B (Cell Signaling) and IRdye 800 rabbit secondary antibody (926-32211; LI-COR Biosciences, Lincoln, NE, USA) as previously described (Cheng et al. 2015) . The primary antibodies are shown in Table 1. Images were collected using the LI-COR Odyssey infrared imager, and densitometry was performed using LI-COR Image Studio Lite, version 5.0 (LI-COR Biosciences).
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