Home Primary Antibodies TRAF6 Antibody [B8B10]

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TRAF6 Antibody [B8B10]

Catalog No.: F4201

    Application: Reactivity:
    • F4201-wb
      Lane 1: K562, Lane 2: Hela, Lane 3: 293T, Lane 4: COS-7

    Usage Information

    Dilution
    1:1000
    1:100
    Application
    WB, IP
    Reactivity
    Human, 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
    60 kDa

    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.

    Datasheet & SDS

    Biological Description

    Specificity
    TRAF6 Antibody [B8B10] detects endogenous levels of total TRAF6 protein. This antibody is not predicted to cross-react with other TRAF family members.
    Subcellular Location
    Cytoplasm, Lipid droplet, Nucleus
    Uniprot ID
    Q9Y4K3
    Clone
    B8B10
    Synonym(s)
    TNF receptor-associated factor 6; E3 ubiquitin-protein ligase TRAF6; Interleukin-1 signal transducer; RING finger protein 85; RING-type E3 ubiquitin transferase TRAF6; TRAF6; RNF85.
    Background
    Tumor necrosis factor receptor–associated factors (TRAFs) constitute a family of cytoplasmic adaptor proteins that mediate diverse intracellular signaling events. In mammals, seven TRAF family members have been identified, including six classical members (TRAF1–TRAF6) and one nonclassical member (TRAF7). The classical TRAFs share a conserved carboxy-terminal TRAF domain, which is absent in the nonclassical TRAF7. Functionally, TRAFs participate in the signal transduction of the tumor necrosis factor superfamily (TNFSF) as well as members of the Toll-like/IL-1 receptor (TLR/ILR) superfamily, thereby regulating downstream signaling cascades such as the mitogen-activated protein kinase (MAPK) pathway. Beyond receptor signaling, TRAFs play essential roles in cellular proliferation, differentiation, apoptosis, and survival, while also modulating immune and inflammatory responses. Dysregulation of TRAF proteins contributes to oncogenesis, with distinct members exerting either oncogenic or tumor-suppressive functions. TRAF1, TRAF2, TRAF4, TRAF5, and TRAF6 have been implicated in carcinogenesis, whereas TRAF3 functions predominantly as a tumor suppressor. Among the family, TRAF6 is distinguished by its unique receptor-binding specificity, enabling it to interact not only with members of the TNF receptor superfamily but also with IL-1R/TLR superfamily proteins. Overexpression of TRAF6 has been reported in multiple tumor types, including colon, gastric, and breast carcinomas, as well as melanoma. Functionally, TRAF6 promotes tumor initiation and progression by modulating apoptosis, proliferation, survival, and invasion. Mechanistically, TRAF6 activates several signaling pathways, with the Toll-like receptor 4 (TLR4) cascade being particularly significant. Through both MyD88-dependent and MyD88-independent routes, TRAF6 amplifies inflammatory and survival signaling. In addition, TRAF6 enhances PI3K–AKT pathway activation by facilitating PI3K ubiquitination, thereby increasing AKT phosphorylation and driving cell growth. Collectively, these findings highlight TRAF6 as a critical adaptor that integrates receptor signaling with oncogenic processes.
    References
    • https://pubmed.ncbi.nlm.nih.gov/32905356/

    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.

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