Home Primary Antibodies CD46 Antibody [H10H10]

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CD46 Antibody [H10H10]

Cat.No.: F2903

    Application: Reactivity:
    • F2903-wb
      Lane 1: HeLa, Lane 2: HeLa (KO CD46), Lane 3: MOLT4, Lane 4: K562

    Usage Information

    Dilution
    1:1000 - 1:10000
    1:500 - 1:1000
    Application
    WB, 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
    44 kDa 50-75 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 normal uterus tissue; Human tonsil tissue; Human colonic adenocarcinoma tissue; Human normal breast tissue; Human kidney tissue; Human normal placenta tissue; HAP1 cells; HeLa cells; K562 cells; MOLT-4 cells; Jurkat cells
    Negative Control

    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.
    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.
     
    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.
    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
    CD46 Antibody [H10H10] detects endogenous levels of total CD46 protein.
    Subcellular Location
    Cytoplasmic vesicle, Membrane
    Uniprot ID
    P15529
    Clone
    H10H10
    Synonym(s)
    CD46; MCP; MIC10; Membrane cofactor protein; TLX; Trophoblast leukocyte common antigen
    Background
    CD46, also known as Membrane Cofactor Protein or MCP, is a ubiquitously expressed type I transmembrane glycoprotein belonging to the regulators of complement activation RCA family, and protects host cells from complement-mediated lysis by serving as a cofactor for factor I-mediated cleavage of C3b and C4b deposited on self-surfaces during classical or alternative pathway activation. Structurally, its extracellular region contains four short consensus repeats SCR1 to SCR4 of about 60 amino acids each, which form compact beta-barrel sushi domains stabilized by three disulfide bonds, followed by a serine-threonine-proline rich O-glycosylated region that confers rigidity, a 12 amino acid spacer, a transmembrane domain, and alternatively spliced cytoplasmic tails, CYT-1 of 16 amino acids with CKII and PKC phosphorylation sites, and CYT-2 of 23 amino acids with Src and CKII sites. SCR2 and SCR3 mediate binding to pathogens, such as HHV-6 gH, gL, gQ1, and gQ2, which linearize the structure, while SCR1 to SCR4 collectively present C3b and C4b for proteolysis. The primary function of CD46 is to maintain innate immune homeostasis by binding fluid-phase or surface-bound C3b and C4b, recruiting factor I to generate iC3b and iC4b as inactivating opsonic fragments, and thereby preventing C3 and C5 convertase amplification and membrane attack complex formation. CD46 also regulates T cell polarization, inducing Tr1 cells via SIRPα and CD9, mediates sperm-oolemma fusion through SCR1 and SCR4, and serves as a receptor for measles virus, HHV-6, Neisseria, and streptococci, facilitating pathogen entry. The CYT-1 and CYT-2 tails signal intracellularly through raft partitioning and calcium flux, and genetic variants that reduce cofactor activity cause atypical hemolytic uremic syndrome aHUS through uncontrolled C3 consumption and thrombotic microangiopathy. Upregulated CD46 shields tumors such as breast, bladder, and medulloblastoma from complement cytotoxicity while promoting migration through increased C3 alpha, MGP, and KRT13.
    References
    • https://pubmed.ncbi.nlm.nih.gov/37840671/
    • https://pubmed.ncbi.nlm.nih.gov/20941397/

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