Home Primary Antibodies Acetyl-p53 (Lys382) Antibody [K2J24]

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Acetyl-p53 (Lys382) Antibody [K2J24]

Catalog No.: F2232

    Application: Reactivity:
    • F2232-wb
      Lane 1: HepG2, Lane 2: HepG2 (starved overnight; etoposide, 30ug/ml, 8 h; Trichostatin A, 500ng/ml, 4 h)

    Experiment Essentials

    WB
    Recommended WB dilution ratio: 1:250
    Exposure time of at least 90s is recommended.

    Usage Information

    Dilution
    1:250 - 1:5000
    1:100 - 1:250
    1:20
    Application
    WB, IF, FCM
    Reactivity
    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
    53 kDa
    Positive Control HepG2 cell (Etoposide, 30ug/ml; Trichostatin A, 500ng/ml); T-47D cell (Etoposide, 30ug/ml; Trichostatin A, 500ng/ml); HeLa cell (Trichostatin A, 500ng/ml)
    Negative Control HepG2 cell

    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 1:250), 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. (Exposure time of at least 90s is recommended)
    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.
     

    Datasheet & SDS

    Biological Description

    Specificity
    Acetyl-p53 (Lys382) Antibody [K2J24] detects endogenous levels of p53 only when acetylated at lysine 382.
    Subcellular Location
    Cytoplasm, Cytoskeleton, Endoplasmic reticulum, Mitochondrion, Nucleus
    Uniprot ID
    P04637
    Clone
    K2J24
    Synonym(s)
    P53; TP53; Cellular tumor antigen p53; Antigen NY-CO-13; Phosphoprotein p53; Tumor suppressor p53
    Background
    Acetyl-p53 (Lys382) refers to the tumor suppressor protein p53 that has undergone acetylation at its conserved carboxyl-terminal lysine 382, a key post-translational modification regulating its stability and activity. Structurally, p53 is a 393-amino acid transcription factor composed of an N-terminal transactivation domain, a central DNA-binding domain, an oligomerization domain, and a C-terminal regulatory domain where Lys382 resides. Acetylation at Lys382, mediated mainly by histone acetyltransferases such as p300/CBP and PCAF, enhances p53’s DNA-binding affinity, prevents MDM2-mediated degradation, and promotes its accumulation in the nucleus. Acetyl-p53 (Lys382) is widely expressed in mammalian cells but its levels are tightly controlled and increase under stress conditions such as DNA damage, oxidative stress, or treatment with histone deacetylase inhibitors (HDACi). Functionally, acetylation at Lys382 not only augments the transcriptional activity of p53 in driving the expression of cell cycle regulators (e.g., p21) and proapoptotic genes (e.g., BAX, PUMA, DR5), but also facilitates its transcription-independent roles at mitochondria. Specifically, Lys382 acetylation enables p53 to disrupt the inhibitory Ku70-BAX complex, thereby freeing BAX to translocate to mitochondria, generate reactive oxygen species, and promote apoptosis. Thus, Acetyl-p53 (Lys382) represents a critical molecular switch integrating stress responses, tumor suppression, and apoptosis.
    References
    • https://pubmed.ncbi.nlm.nih.gov/19265193/

    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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