Home Primary Antibodies TORC1 Antibody [B21M7]

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TORC1 Antibody [B21M7]

Catalog No.: F4063

    Application: Reactivity:
    • F4063-wb
      Lane 1: Mouse brain, Lane 2: Rat brain

    Usage Information

    Dilution
    1:1000 - 1:5000
    1:1000 - 1:10000
    Application
    WB, FCM
    Reactivity
    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
    67 kDa 78 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 Fetal brain; C6 cell; SHSY-5Y cell
    Negative Control

    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: 10%. 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: 200 mA, 120 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.

    Datasheet & SDS

    Biological Description

    Specificity
    TORC1 Antibody [B21M7] detects endogenous levels of total TORC1 protein.
    Subcellular Location
    Cytoplasm, Nucleus
    Uniprot ID
    Q6UUV9
    Clone
    B21M7
    Synonym(s)
    KIAA0616, MECT1, TORC1, WAMTP1, CRTC1, CREB-regulated transcription coactivator 1, Mucoepidermoid carcinoma translocated protein 1, Transducer of regulated cAMP response element-binding protein 1, TORC-1, Transducer of CREB protein 1
    Background
    Target of Rapamycin Complex 1 (TORC1) is a central, multi-protein kinase complex that integrates various signals, such as nutrient, energy, and growth factor availability, to regulate crucial cellular processes like growth, metabolism, and autophagy. TORC1 consists of the serine/threonine kinase mTOR and several regulatory proteins, including the regulatory-associated protein of mTOR (Raptor), mammalian lethal with SEC13 protein 8 (mLST8), PRAS40, and DEPTOR. These components form a dimeric, rhomboid-shaped complex, with mTOR and Raptor playing key roles in dimerization and substrate recruitment. TORC1 drives cellular anabolic processes by phosphorylating downstream targets such as ribosomal protein S6 kinase 1 (S6K1) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), promoting protein synthesis, lipid biogenesis, and nucleotide synthesis, all critical for cell growth and proliferation. TORC1 also suppresses autophagy by phosphorylating ULK1, thus balancing anabolic and catabolic pathways to maintain cellular homeostasis. The activity of TORC1 is tightly regulated, and amino acid availability is sensed through Rag GTPases and the Ragulator complex, which localize TORC1 to the lysosomal surface for activation. Additionally, growth factors activate TORC1 via the PI3K-Akt-TSC-Rheb pathway, while energy stress inhibits TORC1 through AMP-activated protein kinase (AMPK)-mediated phosphorylation of TSC2. PRAS40 acts as an endogenous inhibitor of TORC1, with its inhibitory function being relieved upon phosphorylation.TORC1 can be inhibited by rapamycin pharmacologically by binding to FKBP12 and the FRB domain of mTOR, thereby disrupting the complex’s activity. Hyperactivation of TORC1 is often implicated in various cancers, metabolic disorders, and age-related diseases.
    References
    • https://pubmed.ncbi.nlm.nih.gov/35561748/
    • https://pubmed.ncbi.nlm.nih.gov/20491627/

    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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