Home Primary Antibodies Phospho-CDK1/ CDK2/ CDK3/ CDK5 (Tyr 15) Rabbit mAb

Phospho-CDK1/ CDK2/ CDK3/ CDK5 (Tyr 15) Rabbit mAb

Catalog No.: F2387

    • Lane 1: HeLa
      Lane 2: HeLa (alkaline phosphatase treated)
    1/

    Experiment Essentials

    Subcellular Location: Cytoplasm, Cytoskeleton, Endosome, Nucleus.
    WB
    Recommending using RIPA/NP-40 Lysis Buffer to prepare lysates.
    Recommended wet transfer conditions: 200 mA, 60 min.
    Recommended primary antibody dilution ratio 1:10000.

    Usage Information

    Dilution
    1:10000
    1:100
    1:100
    Application
    WB, IP, IHC
    Source
    Rabbit
    Reactivity
    Human, Rat
    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
    34 kDa, 65 kDa
    Positive Control Human transitional carcinoma of bladder; Rat colon; HeLa; 293T
    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, 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 1:10000), 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
    1249. 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

    Phospho-CDK1/ CDK2/ CDK3/ CDK5 (Tyr 15) Rabbit mAb recognizes endogenous levels of CDK1, CDK2, CDK3 and CDK5 protein only when phosphorylated at Y15.
    Uniprot ID
    P24941,P20248,P24864
    Clone
    B6L9
    Background

    Cyclin-dependent kinases (CDKs) play a pivotal role in various essential cellular processes, including cell cycle regulation, transcription, communication, metabolism, and apoptosis. These kinases operate in a tightly controlled pathway to ensure accurate DNA replication and equal segregation during cell division. Disruption of these processes can lead to apoptosis; however, if left uncorrected, such deregulation may result in diseases like cancer, neurodegenerative disorders, and stroke. 20 members of the CDK family have been identified, each contributing to the regulation of the cell cycle, transcription, and splicing. Among these, cyclin-dependent kinase 1 (CDK1), previously referred to as Cdc2, partners with cyclin B1 to drive the G2-to-mitosis transition. The CDK1/cyclin B1 complex becomes active late in the G2 phase, reaching its peak activity in prometaphase, and remains active until the spindle assembly checkpoint is resolved, allowing the cell to proceed to metaphase. Activation of this complex involves CDC25-mediated dephosphorylation at inhibitory sites Thr14 and Tyr15. In proliferating cells, cyclin-dependent kinase 2 (CDK2) is a key regulator of the G1/S and S/G2 transitions. CDK2, in complex with cyclin E, phosphorylates the retinoblastoma protein (Rb) to initiate S phase entry. Additionally, CDK2 regulates the phosphorylation of various transcription factors and is critical in cell differentiation, proliferation, apoptosis, and the adaptive immune response. Cyclin-dependent kinase 5 (CDK5) shares significant sequence similarity with other CDKs but primarily functions in post-mitotic neurons, where it helps maintain neuronal structure. Unlike other CDKs that pair with cyclins, CDK5 is activated by p35 and p39, regulatory subunits with distinct sequences. Although CDK5 is broadly expressed, its kinase activity is predominantly observed in the nervous system due to the restricted expression of p35 and p39 in post-mitotic neurons. 
    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
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