Home Primary Antibodies Thioredoxin/TRX Rabbit mAb For research use only.

Thioredoxin/TRX Rabbit mAb

Catalog No.: F3590

    Application: Reactivity:

    Experiment Essentials

    WB
    Recommended SDS-PAGE separating gel concentration: 20%.
    Recommended wet transfer conditions: 200 mA, 60 min，Recommended to use 0.22 μm PVDF membrane.
    Recommended WB dilution ratio: 1:10000

    Usage Information

    Dilution
    1:10000-1:50000
    1:10 - 1:100
    1:250 - 1:500
    Application
    WB, IP, IHC
    Reactivity
    Human
    Source
    Rabbit
    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
    12 kDa 12 kDa
    *Why do the predicted and actual molecular weights differ?
    The following reasons may explain differences between the predicted and actual protein molecular weight.

    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: 20%. 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. ( 0.22 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 1:10000), 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
    Thioredoxin/TRX Rabbit mAb recognizes endogenous levels of total Thioredoxin/TRX protein.
    Subcellular Location
    Cytoplasm, Nucleus, Secreted
    Uniprot ID
    P10599
    Clone
    P8C12
    Synonym(s)
    TRDX, TRX1, TXN, ATL-derived factor, Surface-associated sulphydryl protein, ADF, SASP
    Background
    Thioredoxin (Trx) is a highly conserved redox-regulatory protein that plays a central role in maintaining cellular redox homeostasis. It exerts its function by reducing oxidized cysteine residues in target proteins through its characteristic Cys-Gly-Pro-Cys active site motif, which confers its thiol-disulfide oxidoreductase activity. It acts as a direct antioxidant by scavenging reactive oxygen species (ROS) and protecting cells from oxidative stress. It also serves as a cofactor for various enzymes and transcription factors, thereby influencing essential cellular processes such as DNA synthesis, cell proliferation, apoptosis, and inflammation. In cellular senescence, Trx regulates the redox state and modulates redox-sensitive signaling pathways involved in initiating and maintaining senescence. Loss or dysfunction of Trx leads to increased oxidative stress, DNA damage, and activation of key senescence pathways, including the p53-p21 and p16-Rb axes. In the cardiovascular system, Trx is upregulated in response to oxidative and nitrosative stress, where it protects endothelial and vascular smooth muscle cells, promotes tissue repair, and mitigates myocardial injury through both its antioxidant properties and the inhibition of pro-apoptotic signaling molecules such as apoptosis signal-regulating kinase 1 (ASK1). It also serves as a biomarker for aging and age-related diseases, with elevated expression observed in conditions marked by oxidative stress, chronic inflammation, and cardiovascular dysfunction.
    References
    • https://pubmed.ncbi.nlm.nih.gov/38232457/
    • https://pubmed.ncbi.nlm.nih.gov/14645133/

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

    * Indicates a Required Field

    Please enter your name.
    Please enter your email. Please enter a valid email address.
    Please write something to us.