Home Primary Antibodies Phospho-Cyclin D1 (Thr286) Rabbit mAb

Phospho-Cyclin D1 (Thr286) Rabbit mAb

Catalog No.: F0593

    • Lane 1: HT-1080
      Lane 2: HT-1080 (MG132, 10 μM, 4 hours)
    1/

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    Experiment Essentials

    Subcellular Location: Cytoplasm, Membrane, Nucleus.
    WB
    Recommending using RIPA/NP-40 Lysis Buffer to prepare lysates.
    Recommended wet transfer conditions: 200 mA, 60 min.

    Usage Information

    Dilution
    1:1000
    1:50
    1:2000
    1:1600
    Application
    WB, IP, IF, FCM
    Source
    Rabbit
    Reactivity
    Human, Monkey
    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
    36 Kda
    Positive Control HT-1080 (λ phosphatase treated); HT-1080(MG132-treated)
    Negative Control

    Expression & Treatment Conditions

    Sample Treatment Conditions
    HT-1080 λ phosphatase treatment
    HT-1080 MG132 treatment
    Click to view more sample data

    *For predicted expression levels of this protein in various human-derived cells and tissues, please refer to: http://www.proteinatlas.org

    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, Phosphatase Inhibitor Cocktail)，and homogenize the tissue at a low temperature or lyse it by sonication on ice, then incubate on ice for 30 minutes.
    2. Adherent cell: Aspirate the culture medium and transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.
    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.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktail), sonicate to lyse the cells, and incubate on ice for 30 minutes.
    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, 60 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 ( recommending 5% BSA 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
    794. 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:
     
    Sample Preparation
    1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.
    2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.
    3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.
    4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.
     
    Fixation
    1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.
    2. Wash the sample with PBS for 3 times, 3 minutes each time.
     
    Permeabilization
    1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.
    (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)
    Wash the sample with PBS for 3 times, 3 minutes each time.
     
    Blocking
    Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)
    Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.
     
    Immunofluorescence Staining (Day 1)
    1. Remove the blocking solution and add the diluted primary antibody.
    2. Incubate the sample in a humidified chamber at 4°C overnight.
     
    Immunofluorescence Staining (Day 2)
    1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.
    2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.
    3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.
    4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.
    5. Wash with PBST for 3 times, 5 minutes each time.
     
    Mounting
    1. Mount the sample with an anti-fade mounting medium.
    2. Allow the slide to dry at room temperature overnight in the dark.
    3. Store the slide in a slide storage box at 4°C, protected from light.

    Datasheet & SDS

    Biological Description

    Specificity

    Phospho-Cyclin D1 (Thr286) Rabbit mAb detects endogenous levels of cyclin D1 only when phosphorylated at Thr286.
    Uniprot ID
    P24385
    Clone
    P16A10
    Background

    Cyclin D1 is primarily recognized for its nuclear role in regulating cell cycle progression. It controls the transition from the G1 to S phase by acting as an allosteric regulator of cyclin-dependent kinases 4 (CDK4) and 6 (CDK6). The activated Cyclin D1/CDK4 complex translocates to the nucleus, where it phosphorylates the retinoblastoma (RB) protein in conjunction with Cyclin E/CDK2, thereby relieving RB’s inhibitory effect on the E2F transcription factor. This release allows E2F to activate the transcription of genes essential for cell proliferation. In normal cells, Cyclin D1 expression is tightly controlled; however, in cancer, its activity is frequently upregulated through various mechanisms. Dysregulation in Cyclin D1 transcription, accumulation, ubiquitination, and hyperactivation of its associated CDKs contribute to uncontrolled cell growth. As a result, Cyclin D1 is considered an oncogenic driver in several cancers, including breast cancer, lung cancer, and melanoma. Phosphorylation of Cyclin D1 at Thr286 by glycogen synthase kinase 3β or via the Ras/Raf/MEK/MAPK pathway enhances its ubiquitination and subsequent proteasomal degradation.
    References

    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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