Home Primary Antibodies Factor H Antibody [H24L2]

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Factor H Antibody [H24L2]

Catalog No.: F3425

    Application: Reactivity:

    Experiment Essentials

    WB
    Recommended SDS-PAGE separating gel concentration: 5%.

    Usage Information

    Dilution
    1:2000
    Application
    WB, ELISA
    Reactivity
    Human
    Source
    Mouse 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
    139 kDa 170 kDa
    *Why do the predicted and actual molecular weights differ?
    The following reasons may explain differences between the predicted and actual protein molecular weight.
    Positive Control Human serum; Human plasma
    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)，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.
     
    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: 5%. 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.

    Datasheet & SDS

    Biological Description

    Specificity
    Factor H Antibody [H24L2] detects endogenous levels of total Factor H protein.
    Subcellular Location
    Secreted
    Uniprot ID
    P08603
    Clone
    H24L2
    Synonym(s)
    HF; HF1; HF2; CFH; Complement factor H; H factor 1
    Background
    Factor H is a soluble plasma glycoprotein consisting of 20 homologous short consensus repeat (SCR) domains, each about 60 amino acids long, linked linearly to form a flexible elongated molecule. The protein’s N-terminal SCR1-4 domains form the principal regulatory segment responsible for binding the complement fragment C3b and exhibiting decay-accelerating activity toward the alternative pathway C3 convertase (C3bBb). These domains also serve as cofactors for Factor I–mediated cleavage of C3b to inactive fragments, dampening complement activation. SCR6-8 and the C-terminal SCR18-20 domains mediate high-affinity interactions with host polyanionic surfaces such as glycosaminoglycans and sialic acid, enabling Factor H to discriminate self-cells from pathogens and focus complement inhibition locally on host tissues. The middle SCR10-15 domains contribute to protein conformation and weaker interactions with C3 fragments. Factor H inhibits complement activation by competing with Factor B for C3b binding sites, accelerating convertase dissociation, and promoting the proteolytic inactivation of C3b, effectively preventing uncontrolled complement amplification which could damage host tissue. Importantly, certain pathogens hijack Factor H to evade the immune response by binding it to their surfaces. Mutations in the CFH gene or autoantibodies against Factor H lead to diseases such as atypical hemolytic uremic syndrome (aHUS), age-related macular degeneration (AMD), and membranoproliferative glomerulonephritis type II (MPGNII).
    References
    • https://pubmed.ncbi.nlm.nih.gov/18081691/
    • https://pubmed.ncbi.nlm.nih.gov/24970127/

    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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