Immunofluorescent analysis of mTOR (green) in U-2 OS cells. The cells were fixed with formaldehyde for 15 minutes, permeabilized with 0.1% Triton X-100 in TBS for 10 minutes, and blocked with 1% Blocker BSA in PBS (Product # 37525) for 15 minutes, all at room temperature. Cells were stained with an mTOR C-terminal polyclonal antibody (Product # PA1-188) at a dilution of 1:50 in 1% Blocker BSA in PBS (Product # 37525) for 1 hour at room temperature, and then incubated with a DyLight 488-conjugated goat anti-rabbit IgG secondary antibody (Product # 35552) at a dilution of 1:250 for 30 minutes at room temperature. F-Actin (red) was stained with DyLight-554 Phalloidin (Product # 21834) and nuclei (blue) were stained with Hoechst 33342 dye (Product # 62249). Images were taken on a Thermo Scientific ToxInsight Instrument at 20X magnification.
|Tested species reactivity||Human, Non-human primate|
|Host / Isotype||Rabbit / IgG|
|Immunogen||Recombinant protein corresponding to amino acids 2447-2549 of human mTOR expressed in E. coli.|
|Storage buffer||PBS with 30% glycerol, 1mg/ml BSA|
|Contains||0.05% sodium azide|
|Tested Applications||Dilution *|
|Western Blot (WB)||1:2500-1:5000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
Western blot analysis of PA1-188 detects mTOR at ~250 kDa in human and non-human primate cells, slightly below the theoretical MW of ~280kDa. Using a Tris-Acetate SDS gel for high molecular weight protein separation, and a nitrocellulose membrane for protein transfer are necessary for optimal WB results.
Immunofluorescence analysis shows cytoplasmic and some nuclear staining of mTOR in human B3 and U-2 OS cells.
Mechanistic target of rapamycin (mTOR) is a serine/threonine protein kinase which is a central regulator of cellular metabolism, growth, and survival in response to hormones, growth factors, nutrients, energy, and stress signals. mTOR directly or indirectly regulates the phosphorylation of at least 800 proteins, and functions as part of 2 structurally and functionally distinct signaling complexes, mTORC1 and mTORC2 (mTOR complex 1 and 2). Activated mTORC1 up-regulates protein synthesis by phosphorylating key regulators of mRNA translation and ribosome synthesis. As part of the mTORC2 complex, mTOR regulates cellular processes including survival and organization of the cytoskeleton. It plays a critical role in the phosphorylation at Ser-473 of AKT1. Mechanistic target of rapamycin complex 1 (mTORC1) contains mTOR, MLST8, RPTOR, AKT1S1/PRAS40 and DEPTOR. mTORC2 contains MTOR, MLST8, PRR5, RICTOR, MAPKAP1 and DEPTOR. Due to its central role in celliular signaling, mTOR is associated with a vast array of diseases, including many cancers, inflammation, cellular growth, and metabolic disorders.
IP-MS enrichment of MTOR (LFQ intensity): MTOR was enriched 323-fold from HCT116 lysate compared to background proteins, using the optimized IP-MS workflow with Pierce MS-Compatible Magnetic IP Kit protein A/G (Part No. 90409) and MTOR antibody (Part No. PA1-188). See more information on IP-MS verification of antibody selectivity. IP-MS validation info.
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.