Home Primary Antibodies Anti-Akt (pan) Rabbit Antibody [C11A22] For research use only.

Anti-Akt (pan) Rabbit Antibody [C11A22]

Catalog No.: F3511

    Application: Reactivity:

    Usage Information

    Dilution
    1:1000
    1:100
    1:200
    1:100
    1:100
    Application
    WB, IP, IHC, IF, FCM
    Reactivity
    Human, Mouse, Rat, Monkey
    Source
    Rabbit
    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
    60 kDa
    Positive Control Human breast carcinoma; Human lung carcinoma; Human melanoma; AKt1 cell; AKt2 cell; AKt3 cell; C2C12 cell; C6 cell; COS cell; COS-7 cell; 293T cell; NIH/3T3 cell; LNCaP cell; HeLa cell (insulin-treated); Jurkat 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.
    IF
    Experimental Protocol：
     
    Specimen Preparation 
    1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS.
    NOTE: Paraformaldehyde is toxic, use only in a fume hood.
    2. Fix cells for 15 min at room temperature.
    3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
    4. Proceed with Immunostaining.
     
    Immunostaining
    1. Add theblocking buffer and incubate for 60 min at RT.
    2. Prepare primary antibody diluent in antibody dilution buffer as recommended .
    3. Aspirate blocking solution, apply diluted primary antibody.
    4. Incubate overnight at 4°C.
    5. Rinse three times in 1X PBS for 5 min each.
    6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
    7. Rinse three times in 1X PBS for 5 min each.
    8. Mount slides usingmounting medium with DAPI and cover with coverslips.
    9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 23°C protected from light.
     
    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

    Anti-Akt (pan) Rabbit Antibody [C11A22] recognizes endogenous levels of total Akt (pan) protein.
    Subcellular Location
    Cell membrane, Cytoplasm, Endosome, Membrane, Nucleus
    Uniprot ID
    P31751, Q9Y243, P31749
    Clone
    C11A22
    Synonym(s)
    RAC-alpha serine/threonine-protein kinase; Protein kinase B (PKB); Protein kinase B alpha (PKB alpha); Proto-oncogene c-Akt; RAC-PK-alpha; AKT1; PKB; RAC; RAC-gamma serine/threonine-protein kinase; Protein kinase Akt-3; Protein kinase B gamma (PKB gamma); RAC-PK-gamma; STK-2; AKT3; PKBG; RAC-beta serine/threonine-protein kinase; Protein kinase Akt-
    Background
    AKT, also known as protein kinase B (PKB), is a family of three serine/threonine kinases: AKT1, AKT2, and AKT3. These kinases are central regulators of cell survival, growth, proliferation, metabolism, and migration. All AKT isoforms share a conserved structure with an N-terminal pleckstrin homology (PH) domain for membrane targeting, a central kinase domain, and a C-terminal regulatory domain. In the inactive state, the PH domain interacts with the kinase domain to keep AKT in a closed conformation. Activation of the PI3K pathway leads to the production of PIP3, which recruits AKT to the plasma membrane. At the membrane, AKT is phosphorylated by PDK1 at threonine 308 and by mTORC2 at serine 473, resulting in full activation. Activated AKT phosphorylates many substrates involved in cell cycle progression, inhibition of apoptosis, protein synthesis, and metabolic regulation, including mTORC1, GSK-3, BAD, and FOXO transcription factors. AKT1 mainly promotes cell survival and tissue growth. AKT2 is essential for insulin signaling and glucose homeostasis. AKT3 is most abundant in the brain and is important for neuronal development. Although highly similar, the isoforms have non-redundant and sometimes opposing roles, such as AKT1 inhibiting and AKT2 promoting cell migration in some cancers. AKT activity is regulated by membrane recruitment, phosphorylation, and domain interactions, and is negatively regulated by phosphatases like PTEN and PP2A. Dysregulation or hyperactivation of AKT signaling occurs in cancers, diabetes, and other diseases.
    References
    • https://pubmed.ncbi.nlm.nih.gov/39445011/
    • https://pubmed.ncbi.nlm.nih.gov/15784165/

    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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