mTOR Antibody [L10A18]

Catalog No.: F2516

For research use only.

    Home Primary Antibodies mTOR Antibody [L10A18]

    Application: Reactivity:
    • F2516-wb
      Lane 1: MCF7, Lane 2: HepG2, Lane 3: Mouse brain, Lane 4: Rat brain

    Usage Information

    Dilution
    1:1000 - 1:5000
    1:50 - 1:100
    1:400
    Application
    WB, IP, IHC
    Reactivity
    Mouse, Rat, Human
    Source
    Rabbit Monoclonal Antibody
    Storage Buffer
    PBS, pH 7.2+50% Glycerol+0.05% BSA+0.01% NaN3
    Storage (from the date of receipt)
    -20°C (avoid freeze-thaw cycles), 2 years
    Predicted MW Observed MW
    289 kDa 250 kDa
    *Why do the predicted and actual molecular weights differ?
    The following reasons may explain differences between the predicted and actual protein molecular weight.
    Positive Control Human breast carcinoma; Human heart; Rat testis; Rat brain; Mouse testis; Mouse brain; Mouse heart; Mouse thymus; Mouse lung; HeLa cell; A549 cell; HepG2 cell; HEK-293 cell; MCF7 cell; HaCaT cell; MDA-MB-231 cell
    Negative Control Human fetal lung; Rat heart; Rat liver; Rat spleen; Rat thymus; Rat lung; Mouse kidney; Mouse liver

    Exprimental Methods

    WB
    Experimental Protocol:
     
    Sample preparation
    1. Tissue: Lyse the tissue sample by adding an appropriate volume of ice-cold RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail)，and homogenize the tissue at a low temperature.
    2. Adherent cell: Aspirate the culture medium and wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.
    3. Suspension cell: Transfer the culture medium to a pre-cooled centrifuge tube. Centrifuge and aspirate the supernatant. Wash the cells with ice-cold PBS twice. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail) and put the sample on ice for 5 min.
    4. Place the lysate into a pre-cooled microcentrifuge tube. Centrifuge at 4°C for 15 min. Collect the supernatant;
    5. Remove a small volume of lysate to determine the protein concentration;
    6. Combine the lysate with protein loading buffer. Boil 20 µL sample under 95-100°C for 5 min. Centrifuge for 5 min after cool down on ice.
     
    Electrophoretic separation
    1. According to the concentration of extracted protein, load appropriate amount of protein sample and marker onto SDS-PAGE gels for electrophoresis. Recommended separating gel (lower gel) concentration: 5%. Reference Table for Selecting SDS-PAGE Separation Gel Concentrations
    2. Power up 80V for 30 minutes. Then the power supply is adjusted (110 V~150 V), the Marker is observed, and the electrophoresis can be stopped when the indicator band of the predyed protein Marker where the protein is located is properly separated. (Note that the current should not be too large when electrophoresis, too large current (more than 150 mA) will cause the temperature to rise, affecting the result of running glue. If high currents cannot be avoided, an ice bath can be used to cool the bath.)
     
    Transfer membrane
    1. Take out the converter, soak the clip and consumables in the pre-cooled converter;
    2. Activate PVDF membrane with methanol for 1 min and rinse with transfer buffer;
    3. Install it in the order of "black edge of clip - sponge - filter paper - filter paper - glue -PVDF membrane - filter paper - filter paper - sponge - white edge of clip";
    4. The protein was electrotransferred to PVDF membrane. ( 0.45 µm PVDF membrane is recommended ) Reference Table for Selecting PVDF Membrane Pore Size Specifications
    Recommended conditions for wet transfer: 250 mA, 180 min.
    ( Note that the transfer conditions can be adjusted according to the protein size. For high-molecular-weight proteins, a higher current and longer transfer time are recommended. However, ensure that the transfer tank remains at a low temperature to prevent gel melting.)
     
    Block
    1. After electrotransfer, wash the film with TBST at room temperature for 5 minutes;
    2. Incubate the film in the blocking solution for 1 hour at room temperature;
    3. Wash the film with TBST for 3 times, 5 minutes each time.
     
    Antibody incubation
    1. Use 5% skim milk powder to prepare the primary antibody working liquid (recommended dilution ratio for primary antibody 1:1000), gently shake and incubate with the film at 4°C overnight;
    2. Wash the film with TBST 3 times, 5 minutes each time;
    3. Add the secondary antibody to the blocking solution and incubate with the film gently at room temperature for 1 hour;
    4. After incubation, wash the film with TBST 3 times for 5 minutes each time.
     
    Antibody staining
    1. Add the prepared ECL luminescent substrate (or select other color developing substrate according to the second antibody) and mix evenly;
    2. Incubate with the film for 1 minute, remove excess substrate (keep the film moist), wrap with plastic film, and expose in the imaging system.
    IHC
    Experimental Protocol：
     
    Deparaffinization/Rehydration
    1. Deparaffinize/hydrate sections:
    2. Incubate sections in three washes of xylene for 5 min each.
    3. Incubate sections in two washes of 100% ethanol for 10 min each.
    4. Incubate sections in two washes of 95% ethanol for 10 min each.
    5. Wash sections two times in dH2O for 5 min each.
    6.Antigen retrieval: For Citrate: Heat slides in a microwave submersed in 1X citrate unmasking solution until boiling is initiated; continue with 10 min at a sub-boiling temperature (95°-98°C). Cool slides on bench top for 30 min.
     
    Staining
    1. Wash sections in dH2O three times for 5 min each.
    2. Incubate sections in 3% hydrogen peroxide for 10 min.
    3. Wash sections in dH2O two times for 5 min each.
    4. Wash sections in wash buffer for 5 min.
    5. Block each section with 100–400 µl of blocking solution for 1 hr at room temperature.
    6. Remove blocking solution and add 100–400 µl primary antibody diluent in to each section. Incubate overnight at 4°C.
    7. Remove antibody solution and wash sections with wash buffer three times for 5 min each.
    8. Cover section with 1–3 drops HRPas needed. Incubate in a humidified chamber for 30 min at room temperature.
    9. Wash sections three times with wash buffer for 5 min each.
    10. Add DAB Chromogen Concentrate to DAB Diluent and mix well before use.
    11. Apply 100–400 µl DAB to each section and monitor closely. 1–10 min generally provides an acceptable staining intensity.
    12. Immerse slides in dH2O.
    13. If desired, counterstain sections with hematoxylin.
    14. Wash sections in dH2O two times for 5 min each.
    15. Dehydrate sections: Incubate sections in 95% ethanol two times for 10 sec each; Repeat in 100% ethanol, incubating sections two times for 10 sec each; Repeat in xylene, incubating sections two times for 10 sec each.
    16. Mount sections with coverslips and mounting medium.
     

    Datasheet & SDS

    Biological Description

    Specificity
    mTOR Antibody [L10A18] detects endogenous levels of total mTOR protein.
    Uniprot ID
    P42345
    Clone
    L10A18
    Synonym(s)
    FRAP, FRAP1, FRAP2, RAFT1, RAPT1, MTOR, Serine/threonine-protein kinase mTOR, FK506-binding protein 12-rapamycin complex-associated protein 1, FKBP12-rapamycin complex-associated protein, Mammalian target of rapamycin, Mechanistic target of rapamycin, Rapamycin and FKBP12 target 1, Rapamycin target protein 1, mTOR
    Background
    mTOR, a serine/threonine kinase belonging to the PI3K-related kinase (PIKK) family, serves as the catalytic core of two distinct complexes: mTOR Complex 1 (mTORC1) and mTOR Complex 2 (mTORC2). mTORC1 is composed of three essential subunits—mTOR, Raptor (regulatory-associated protein of mTOR), and mLST8 (mammalian lethal with Sec13 protein 8, also known as GβL). For cells to grow and proliferate, they must enhance the synthesis of proteins, lipids, and nucleotides while concurrently inhibiting catabolic pathways such as autophagy. mTORC1 orchestrates this balance by integrating environmental cues to regulate anabolic and catabolic processes. One of the primary roles of mTORC1 is to stimulate protein synthesis. It does so by phosphorylating two major downstream effectors: p70 S6 Kinase 1 (S6K1) and eIF4E Binding Protein (4EBP). Specifically, mTORC1 phosphorylates S6K1 at Thr389 within its hydrophobic motif, enabling subsequent activation by PDK1. Beyond protein synthesis, mTORC1 promotes cellular growth by actively repressing autophagy and other catabolic pathways. In contrast, mTORC2 primarily regulates proliferation and survival by phosphorylating members of the AGC kinase family (PKA, PKG, and PKC). Its most critical function is the phosphorylation and activation of Akt, a central effector of insulin/PI3K signaling. Activated Akt drives survival, growth, and proliferation by phosphorylating and inhibiting key substrates, including FoxO1/3a transcription factors, the metabolic regulator GSK3β, and TSC2, an upstream inhibitor of mTORC1. Additionally, mTORC2 activates SGK1, another AGC kinase that influences ion transport and cell survival.
    References
    • https://pubmed.ncbi.nlm.nih.gov/28283069/

    Tech Support

    Answers to questions you may have can be found in the inhibitor handling instructions. Topics include how to prepare stock solutions, how to store inhibitors, and issues that need special attention for cell-based assays and animal experiments.

    Handling Instructions

    Tel: +1-832-582-8158 Ext:3
    If you have any other enquiries, please leave a message.

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