Anti-Phospho-mTOR (Ser2481) Polyclonal Antibody is a Rabbit antibody targeting Phospho-mTOR (Ser2481). Anti-Phospho-mTOR (Ser2481) Polyclonal Antibody can be used in FCM,ICC/IF,IF,IHC-Fr,IHC-P,WB.

IgG

Human,Mouse,Rat (predicted:Dog,Pig,Horse,Rabbit,Sheep)

1. Blank control: A431. Primary Antibody (green line): Rabbit Anti-Phospho-mTOR (Ser2481) antibody (TMAB-01459)
Dilution: 1 μg/Test;
Secondary Antibody: Goat anti-rabbit IgG-AF488
Dilution: 0.5 μg/Test.
Protocol
The cells were fixed with 4% PFA (10 min at room temperature) and then permeabilized with 90% ice-cold methanol for 20 min at-20°C. The cells were then incubated in 5% BSA to block non-specific protein-protein interactions for 30 min at room temperature. Cells stained with Primary Antibody for 30 min at room temperature. The secondary antibody used for 40 min at room temperature.
2. A431 cell; 4% Paraformaldehyde-fixed; Triton X-100 at room temperature for 20 min; Blocking buffer (normal goat serum) at 37°C for 20 min; Antibody incubation with (Phospho-mTOR (Ser2481)) polyclonal Antibody, Unconjugated (TMAB-01459) 1:100, 90 minutes at 37°C; followed by a conjugated Goat Anti-Rabbit IgG antibody at 37°C for 90 minutes, DAPI (blue) was used to stain the cell nucleus.
3. Sample:
Hepg2 (Human) Cell Lysate at 30 μg
Primary: Anti-Phospho-mTOR (Ser2481) (TMAB-01459) at 1/300 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 289 kDa
Observed band size: 289 kDa
4. Tissue/cell: rat brain tissue; 4% Paraformaldehyde-fixed and paraffin-embedded;
Antigen retrieval: citrate buffer (0.01M, pH6.0), Boiling bathing for 15 min; Block endogenous peroxidase by 3% Hydrogen peroxide for 30 min; Blocking buffer (normal goat serum) at 37°C for 20 min;
Incubation: Anti-Phospho-mTOR (Ser2481) Polyclonal Antibody, Unconjugated (TMAB-01459) 1:200, overnight at 4°C, followed by conjugation to the secondary antibody and DAb staining.
5. Paraformaldehyde-fixed, paraffin embedded (mouse brain tissue); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (Phospho-mTOR (Ser2481)) Polyclonal Antibody, Unconjugated (TMAB-01459) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
6. Blank control: mouse splenocytes (blue)
Isotype Control Antibody: Rabbit Igg (orange);
Secondary Antibody: Goat anti-rabbit IgG-FITC (white blue),
Dilution: 1:100 in 1 X PBS containing 0.5% BSA;
Primary Antibody Dilution: 5 μL in 100 μL 1X PBS containing 0.5% BSA (green).
7. Blank control (blue line): Hela (fixed with 70% ethanol (Overnight at 4°C) and then permeabilized with 90% ice-cold methanol for 30 min on ice)
Primary Antibody (green line): Rabbit Anti-Phospho-mTOR (Ser2481) antibody (TMAB-01459), Dilution: 1 μg/10^6 cells;
Isotype Control Antibody (orange line): Rabbit IgG.
Secondary Antibody (white blue line): Goat anti-rabbit IgG-FITC, Dilution: 1 μg/test.
8. Paraformaldehyde-fixed, paraffin embedded (rat brain); Antigen retrieval by boiling in sodium citrate buffer (pH6.0) for 15 min; Block endogenous peroxidase by 3% hydrogen peroxide for 20 min; Blocking buffer (normal goat serum) at 37°C for 30 min; Antibody incubation with (Phospho-mTOR (Ser2481)) Polyclonal Antibody, Unconjugated (TMAB-01459) at 1:200 overnight at 4°C, followed by operating according to SP Kit (Rabbit) instructionsand DAB staining.
9. Sample:
Jurkat (Human) Cell Lysate at 30 μg
MCF-7 (Human) Cell Lysate at 30 μg
Primary: Anti-Phospho-mTOR (Ser2481) (TMAB-01459) at 1/1000 dilution
Secondary: IRDye800CW Goat Anti-Rabbit IgG at 1/20000 dilution
Predicted band size: 289 kDa
Observed band size: 289 kDa

WB: 1:500-2000; IHC-P: 1:100-500; IHC-Fr: 1:100-500; ICC/IF: 1:100; IF: 1:100-500; FCM: 1μg /test

Polyclonal

KLH conjugated Synthesised phosphopeptide: human mTOR around the phosphorylation site of Ser2481

Human

MTOR

Serine/threonine-protein kinase mTOR

Cell cycle inhibitors,Transcription factors/regulators,Cell Cycle Inhibitors,DNA Damage Recognition,Growth factors and hormones ELISA kits,Obesity

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. 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. This includes phosphorylation of EIF4EBP1 and release of its inhibition toward the elongation initiation factor 4E (eiF4E). Moreover, phosphorylates and activates RPS6KB1 and RPS6KB2 that promote protein synthesis by modulating the activity of their downstream targets including ribosomal protein S6, eukaryotic translation initiation factor EIF4B and the inhibitor of translation initiation PDCD4. Regulates ribosome synthesis by activating RNA polymerase III-dependent transcription through phosphorylation and inhibition of MAF1 a RNA polymerase III-repressor. In parallel to protein synthesis, also regulates lipid synthesis through SREBF1/SREBP1 and LPIN1. To maintain energy homeostasis mTORC1 may also regulate mitochondrial biogenesis through regulation of PPARGC1A. mTORC1 also negatively regulates autophagy through phosphorylation of ULK1. Under nutrient sufficiency, phosphorylates ULK1 at 'Ser-758', disrupting the interaction with AMPK and preventing activation of ULK1. Also prevents autophagy through phosphorylation of the autophagy inhibitor DAP. mTORC1 exerts a feedback control on upstream growth factor signaling that includes phosphorylation and activation of GRB10 a INSR-dependent signaling suppressor. Among other potential targets mTORC1 may phosphorylate CLIP1 and regulate microtubules. As part of the mTORC2 complex MTOR may regulate other cellular processes including survival and organization of the cytoskeleton. Plays a critical role in the phosphorylation at 'Ser-473' of AKT1, a pro-survival effector of phosphoinositide 3-kinase, facilitating its activation by PDK1. mTORC2 may regulate the actin cytoskeleton, through phosphorylation of PRKCA, PXN and activation of the Rho-type guanine nucleotide exchange factors RHOA and RAC1A or RAC1B. mTORC2 also regulates the phosphorylation of SGK1 at 'Ser-422'.