Home Primary Antibodies C Reactive Protein Antibody [N20E9]

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C Reactive Protein Antibody (Rabbit mAb) [N20E9]

Cat.No.: F9881

    Application: Reactivity:
    • F9881-wb
      Lane 1: Rat liver, Lane 2: Rat kidney, Lane 3: Mouse plasma

    Experiment Essentials

    WB
    Recommended wet transfer conditions: 200 mA, 60 min.

    Usage Information

    Dilution
    1:1000
    1:30
    1:2000
    Application
    WB, IP, IHC
    Reactivity
    Mouse, Rat, 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
    25 kDa 27 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 liver tissue; Human kidney tissue; Rat liver tissue; Rat kidney tissue; Mouse liver tissue; Human plasma; Mouse plasma; Mouse serum
    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 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. 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 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
    C Reactive Protein Antibody (Rabbit mAb) [N20E9] detects endogenous levels of total C Reactive Protein protein.
    Subcellular Location
    Secreted
    Uniprot ID
    P02741
    Clone
    N20E9
    Synonym(s)
    PTX1, CRP, C-reactive protein
    Background
    C‑reactive protein (CRP) is a highly conserved pentraxin-family acute‑phase protein synthesized predominantly by hepatocytes under transcriptional control of IL‑6, IL‑1β, and TNF, where it is secreted as a cyclic pentamer that circulates in plasma and acts as a pattern‑recognition molecule linking tissue injury and infection to complement activation and opsonophagocytic clearance. The native pentameric structure arranges five identical noncovalently associated subunits around a central pore, each subunit forming a β‑sandwich fold with a Ca²⁺‑dependent ligand‑binding site that recognizes phosphocholine, oxidized phospholipids, and neoepitopes on apoptotic cells, nuclear particles, and some bacterial surfaces; this modular architecture allows CRP to bind a wide spectrum of damage‑ and pathogen‑associated ligands with high avidity. Ligand‑bound CRP efficiently engages C1q to initiate the classical complement cascade and interacts with Fcγ receptors on phagocytes, which together promote opsonization, complement deposition, and phagocytic uptake of apoptotic bodies, necrotic debris, and microbes, and also drive local production of pro‑inflammatory cytokines that amplify the acute‑phase response. In vascular tissues and atheromatous plaques, CRP is detected in both its native pentameric and dissociated monomeric forms, and experimental data indicate that CRP can contribute directly to atherogenesis by promoting endothelial activation, monocyte recruitment, uptake of modified lipids, smooth muscle cell apoptosis, and thrombogenic changes, in addition to reflecting upstream inflammatory signaling. High‑sensitivity CRP measurement in serum provides a stable, reproducible index of low‑grade systemic inflammation, and large prospective cohort studies show that baseline CRP levels in otherwise healthy individuals independently predict future risk of myocardial infarction, stroke, peripheral arterial disease, and sudden cardiac death across a wide range of traditional risk factor profiles, with predictive power that is at least comparable to, and additive with, LDL cholesterol and Framingham risk scores. Elevated CRP also tracks with disease activity and joint damage in rheumatoid arthritis and other chronic inflammatory disorders.
    References
    • https://pubmed.ncbi.nlm.nih.gov/27433484/
    • https://pubmed.ncbi.nlm.nih.gov/12551853/

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