Phospho-mTOR (Ser2448) Monoclonal Antibody (MRRBY), PerCP-eFluor™ 710, eBioscience™

Product Specific Information

Description: This MRRBY monoclonal antibody recognizes human and mouse mammalian target of rapamycin (also known as mTOR, FRAP, RAFT) when phosphorylated on S2448. mTOR is a serine/threonine protein kinase that functions as an ATP and amino acid sensor as well as to balance nutrient availability with cell growth, proliferation, motility, survival, protein synthesis, and transcription. Activated mTOR increases production of enzymes necessary for glycolysis and controls the uptake of glucose and other nutrients. Increased glucose uptake and metabolism helps fulfill the energy needs for mTOR-driven cell growth and proliferation. When sufficient nutrients are available, mTOR transmits a positive signal to p70 S6 kinase and participates in the inactivation of the eIF4E inhibitor, 4E-BP1. mTOR is phosphorylated at S2448 via the PI3 kinase/Akt signaling pathway and is autophosphorylated at Ser2481. Due to its critical role in regulation of cell growth, survival, and metabolism, and because it is often abnormally regulated in tumors, mTOR is under investigation as a potential target for anti-cancer therapy.

Applications Reported: This MRRBY antibody has been reported for use in intracellular staining followed by flow cytometric analysis.

Applications Tested: This MRRBY antibody has been pre-titrated and tested by intracellular staining followed by flow cytometric analysis of normal human peripheral blood cells. This can be used at 5 µL (0.125 µg) per test. A test is defined as the amount (µg) of antibody that will stain a cell sample in a final volume of 100 µL. Cell number should be determined empirically but can range from 10^5 to 10^8 cells/test.

Staining Protocol: All protocols work well for this monoclonal antibody. Use of Protocol A: Two-step protocol: intracellular (cytoplasmic) proteins allows for the greatest flexibility for detection of surface and intracellular (cytoplasmic) proteins. Use of Protocol B: One-step protocol: intracellular (nuclear) proteins is recommended for staining of transcription factors in conjunction with surface and phosphorylated intracellular (cytoplasmic) proteins. Protocol C: Two-step protocol: Fixation/Methanol allows for the greatest discrimination of phospho-specific signaling between unstimulated and stimulated samples, but with limitations on the ability to stain specific surface proteins (refer to "Clone Performance Following Fixation/Permeabilization" located in the BestProtocols Section under the Resources tab online). All Protocols can be found in the Flow Cytometry Protocols: "Staining Intracellular Antigens for Flow Cytometry Protocol" located in the BestProtocols® Section under the Resources tab online.

PerCP-eFluor® 710 emits at 710 nm and is excited with the blue laser (488 nm); it can be used in place of PerCP-Cyanine5.5. We recommend using a 710/50 bandpass filter, however, the 695/40 bandpass filter is an acceptable alternative. Please make sure that your instrument is capable of detecting this fluorochrome.

Light sensitivity: This tandem dye is sensitive to photo-induced oxidation. Please protect this vial and stained samples from light.

Fixation: Samples can be stored in IC Fixation Buffer (Product # 00-8222) (100 µL of cell sample + 100 µL of IC Fixation Buffer) or 1-step Fix/Lyse Solution (Product # 00-5333) for up to 3 days in the dark at 4°C with minimal impact on brightness and FRET efficiency/compensation. Some generalizations regarding fluorophore performance after fixation can be made, but clone specific performance should be determined empirically.

Excitation: 488 nm; Emission: 710 nm; Laser: Blue Laser.

Filtration: 0.2 µm post-manufacturing filtered.

Target Information

FRAP1 (mTOR) is a serine/threonine kinase that plays a critical role in cellular growth and proliferation. Perturbations in the mTOR/PI3-kinase/AKT pathway are associated with numerous forms of cancer. FRAP1 is also the target of rapamycin and its analogues, which are currently used as immunosuppressants and cancer therapeutics. Mutations affecting the gene results in Smith-Kingsmore syndrome.