Home Primary Antibodies SDHA Antibody [D7C19]

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SDHA Antibody [D7C19]

Catalog No.: F4217

    Application: Reactivity:
    • F4217-wb
      Lane 1: Hela, Lane 2: DLD-1, Lane 3: MCF7, Lane 4: C2C12

    Usage Information

    Dilution
    1:1000
    1:50
    1:100 - 1:400
    1:50 - 1:200
    1:50 - 1:200
    Application
    WB, IP,IHC, IF, FCM
    Reactivity
    Human, Mouse, Rat, Hamster, Monkey
    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
    70 kDa
    Positive Control Human colon carcinoma; HeLa cell; DLD-1 cell; MCF-7 cell; C2C12 cell; KNRK cell; NBT-II cell; COS-7 cell; CHO cell
    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: 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 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.
    IF
    Experimental Protocol：
     
    Specimen Preparation 
    1. Aspirate liquid, then cover cells to a depth of 2–3 mm with 4% Paraformaldehyde diluted in 1X PBS.
    NOTE: Paraformaldehyde is toxic, use only in a fume hood.
    2. Fix cells for 15 min at room temperature.
    3. Aspirate fixative, rinse three times in 1X PBS for 5 min each.
    4. Proceed with Immunostaining.
     
    Immunostaining
    1. Add theblocking buffer and incubate for 60 min at RT.
    2. Prepare primary antibody diluent in antibody dilution buffer as recommended .
    3. Aspirate blocking solution, apply diluted primary antibody.
    4. Incubate overnight at 4°C.
    5. Rinse three times in 1X PBS for 5 min each.
    6. Incubate specimens in fluorochrome-conjugated secondary antibody diluted in antibody dilution buffer for 1–2 hr at room temperature in the dark.
    7. Rinse three times in 1X PBS for 5 min each.
    8. Mount slides usingmounting medium with DAPI and cover with coverslips.
    9. For best results, allow mountant to cure overnight at room temperature. For long-term storage, store slides flat at 23°C protected from light.
     
    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
    SDHA Antibody [D7C19] detects endogenous levels of total SDHA protein.
    Subcellular Location
    Membrane, Mitochondrion, Mitochondrion inner membrane
    Uniprot ID
    P31040
    Clone
    D7C19
    Synonym(s)
    Succinate dehydrogenase [ubiquinone] flavoprotein subunit; mitochondrial; Flavoprotein subunit of complex II (Fp); Malate dehydrogenase [quinone] flavoprotein subunit; SDHA; SDH2; SDHF.
    Background
    Succinate dehydrogenase (SDH), also referred to as mitochondrial complex II, is a dual-function enzyme complex that participates in both the tricarboxylic acid (TCA) cycle and the electron transport chain (ETC). It consists of four subunits: SDHA, SDHB, SDHC, and SDHD. SDHA, the flavoprotein catalytic subunit, is a 664–amino acid protein of approximately 72.7 kDa. It contains an FAD-binding domain required for succinate oxidation, along with capping and helical domains that provide structural stability. Ubiquitously expressed and enriched in metabolically active tissues such as the heart and brain, SDHA catalyzes the conversion of succinate to fumarate while transferring electrons to ubiquinone, thereby coupling the TCA cycle to oxidative phosphorylation. Beyond its metabolic role, SDHA also functions as a tumor suppressor. Pathogenic variants in SDHA are associated with mitochondrial disorders such as Leigh syndrome and with tumorigenesis, including gastrointestinal stromal tumors (GISTs) and hereditary paragangliomas. Mutations in the other SDH subunits—SDHB, SDHC, and SDHD—as well as in SDHAF2, which is required for FAD incorporation into SDHA, underlie pheochromocytoma-paraganglioma syndromes. These findings underscore the essential role of SDH not only in energy metabolism but also in maintaining cellular homeostasis and preventing oncogenic transformation.
    References
    • https://pubmed.ncbi.nlm.nih.gov/21752896/
    • https://pubmed.ncbi.nlm.nih.gov/20484225/

    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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