Home Primary Antibodies CD39 Antibody (Rabbit mAb) [A7P6]

research use only

CD39 Antibody (Rabbit mAb) [A7P6]

CatNo: F2610

    Application: Reactivity:
    • F2610-wb
      Lane 1: IM-9

    Usage Information

    Dilution
    1:1000
    1:30
    1:2000
    Application
    WB, IP, IHC
    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 Observed MW
    58 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.
    Post-translational modifications(e.g., phosphorylation, glycosylation); Splice variants and isoforms; Relative charge; Multimerization.
    Positive Control Human pancreas tissue; Human endometrial carcinoma tissue; Human liver tissue; Human placenta tissue; Human heart tissue; IM-9 cells; Human PBMC cells; THP-1 cells
    Negative Control Jurkat cells

    Experimental 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 or lyse it by sonication on ice, then incubate on ice for 30 minutes.
    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) , 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. Lyse the cells by adding an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease 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. Add protein loading buffer to the 20 μL sample, and keep it on ice for immediate use; or determine the optimal denaturation conditions by boiling the sample at a temperature gradient (e.g., 37°C, 50°C, 70°C, 90°C, and 100°C). Cool the sample on ice and centrifuge for 5 min.
     
    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 primary antibody dilution buffer 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.
    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
    CD39 Antibody (Rabbit mAb) [A7P6] detects endogenous levels of total CD39 protein.
    Subcellular Location
    Membrane
    Uniprot ID
    P49961
    Clone
    A7P6
    Synonym(s)
    CD39, ENTPD1, ATP diphosphohydrolase, Ecto-ATP diphosphohydrolase 1, Ecto-apyrase, Lymphoid cell activation antigen, ATP-DPH, ATPDase, Ecto-ATPDase 1, Ecto-ATPase 1, NTPDase1
    Background
    CD39 (ENTPD1) is a prototypic ectonucleoside triphosphate diphosphohydrolase of the E‑NTPDase family that is anchored in the plasma membrane with two short transmembrane segments flanking a large extracellular catalytic loop, where it sequentially hydrolyzes extracellular nucleoside triphosphates and diphosphates, such as ATP and ADP, to AMP and thereby calibrates the magnitude, duration and chemical nature of purinergic signals in the vascular and immune systems. The extracellular domain contains the conserved apyrase signature motifs and metal‑binding residues that coordinate divalent cations and the γ‑ and β‑phosphates of ATP or ADP, allowing CD39 to remove two phosphate groups in successive steps and convert pro‑inflammatory, platelet‑activating ATP/ADP into AMP, which is subsequently dephosphorylated by the ecto‑5′‑nucleotidase CD73 to generate adenosine; in this way, CD39 functions as the rate‑limiting enzymatic step in the ATP→ADP→AMP→adenosine cascade. At the vascular interface, CD39 expressed on endothelial cells and platelets controls thromboregulation by hydrolyzing ATP and ADP in the extracellular space, blocking P2Y receptor–mediated platelet aggregation, limiting microvascular thrombosis and supporting blood flow, and genetic or pharmacologic disruption of CD39 activity increases susceptibility to thrombosis and ischemia–reperfusion injury, highlighting its importance for hemostatic balance. In immunity, CD39 is broadly expressed on B cells, dendritic cells, monocytes, macrophages, neutrophils, activated effector T cells and regulatory T cells, and its phosphohydrolytic activity shapes T‑cell activation, polarization and stability by scavenging pro‑inflammatory ATP released during inflammation, hypoxia or cell damage and facilitating the accumulation of immunosuppressive adenosine via CD73, thus shifting the environment from ATP‑driven P2 receptor signaling (calcium mobilization, chemotaxis, inflammasome activation) toward P1 receptor–mediated anti‑inflammatory signals. CD39 expression on conventional and regulatory T cells is dynamically regulated by cytokines and microenvironmental cues, and CD39⁺ Tregs show enhanced capacity to suppress effector responses in autoimmunity and transplantation through combined ATP degradation and adenosine generation, while high CD39 levels in inflamed tissues confer a competitive advantage for T‑cell–mediated immunoregulation where extracellular ATP concentrations are elevated. In cancer, CD39 is upregulated on intratumoral T cells, myeloid cells and sometimes tumor cells themselves, and reviews emphasize that CD39, together with CD73, drives the conversion of immunostimulatory ATP released by dying cells into immunosuppressive adenosine, establishing an adenosine‑rich tumor microenvironment that dampens cytotoxic T‑cell and NK‑cell function via A2A and A2B receptors and supports tumor progression; CD39 is therefore regarded as an immunological switch and emerging checkpoint target, with therapeutic blockade of CD39 proposed to enhance antitumor immunity by preserving ATP signaling and reducing adenosine accumulation. CD39 dysregulation has been linked to a spectrum of immune‑related disorders, including inflammatory bowel disease, sepsis, multiple sclerosis, allergic disease, systemic lupus erythematosus, diabetes and vascular inflammatory states, reflecting its central position at the crossroads between extracellular nucleotide metabolism, thrombosis and inflammation.
    References
    • https://pubmed.ncbi.nlm.nih.gov/27236363/
    • https://pubmed.ncbi.nlm.nih.gov/23601906/

    Tech Support

    Handling Instructions

    Tel: +1-832-582-8158 Ext:3

    If you have any other enquiries, please leave a message.