ホームページ 一次抗体 Phospho-IKB α (Ser36) Antibody [B13N13] For research use only.
Not for use in humans.

Phospho-IKB α (Ser36) Antibody [B13N13]

Catalog No.: F2237

    異なるサンプルでの発現パターンについては、“発現量と処理条件の例”を参照してください。
    Application: Reactivity:
    • Lane 1: C6 , Lane 2: C6 (Calyculin A, 100ng/ml ,60 min) , Lane 3: C6 (Calyculin A, 100ng/ml ,60 min; Alkaline phosphatase-treated)
    1/
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    文献中Selleckの製品使用例(1)

    カスタマーフィードバック(2)

    キーポイント

    WB
    転写条件（ウェット）: 200 mA, 60 min。
    推奨WB希釈率: 1:10000

    C6などの特定の細胞株では、IKB αのSer 36のリン酸化レベルを高く維持するために、外因性刺激（24時間飢餓状態にした後、100 ng /mLのCalyculin Aで60分間処理）を加えることが推奨されます。
    組織サンプルの場合、最適なローディング量は、ローディング勾配前実験によって最適化する必要があります。
    サンプルタンパク質のリン酸化修飾と完全性を維持するために、サンプル調製中にプロテアーゼ阻害剤とホスファターゼ阻害剤を加えることが推奨されます。
    偽陰性結果を除外するために、翻訳後修飾されたIKB αタンパク質検出を実行しながら、総IKB αタンパク質レベルを定量することが推奨されます。

    使用情報

    Dilution
    1:10000
    1:100
    Application
    WB, FCM
    Source
    Rabbit Monoclonal Antibody
    Reactivity
    Human, Mouse, Rat
    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
    35-40 kDa
    ポジティブコントロール NIH/3T3 (starved for 24 h, then treated with 100nM Calyculin A for 30 min); C6 (starved for 24 h, then treated with 100ng /mL Calyculin A for 60 min); THP-1; HeLa
    ネガティブコントロール NIH/3T3 (starved for 24 h); C6

    サンプル処理データの例

    Cell Line Expression Level References Sample Treatment
    BV2 High Expression 1μg/mL LPS 24h
    クリックして、さらに多くのサンプルデータを表示

    *異なるヒト由来細胞や組織における発現量の予測については、以下をご参照ください: http://www.proteinatlas.org

    プロトコール

    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, Phosphatase 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 transfer the cells into an EP tube. Wash the cells with ice-cold PBS twice. Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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.Add an appropriate volume of RIPA/NP-40 Lysis Buffer (containing Protease Inhibitor Cocktail, Phosphatase 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 ( recommending 5% BSA 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
    1126. 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

    生物学的記述

    Specificity

    Phospho-IKB α (Ser36) Antibody [B13N13] recognizes endogenous levels of total IKBα protein when phosphorylated at Ser 36.
    タンパク質の局在
    細胞質、细胞核
    Uniprot ID
    P25963
    Clone
    B13N13
    Background

    Nuclear factor-kappa B (NF-κB) is a key family of transcription regulators within the NF-κB/Rel protein family, present in nearly all cell types. Its activity is tightly controlled by the inhibitor protein IκB, which binds to NF-κB dimers—either homologous or heterologous—forming a trimeric complex that keeps NF-κB sequestered and inactive in the cytoplasm. IκBα, the most prevalent member of the IκB family, has the ability to shuttle between the cytoplasm and nucleus, leading to a dynamic intracellular distribution. The human IκBα protein consists of 317 amino acids and has a molecular weight of 36 kDa. In its free form, IκBα exhibits a molten globule structure. Its N-terminal signal response domain (SRD) plays a crucial role in receiving phosphorylation, ubiquitination, and SUMOylation signals. This domain contains phosphorylation sites at Ser32, Ser36, and Tyr42, ubiquitination sites at Lys21 and Lys22, and SUMOylation sites at Lys21 and Lys38. The SRD is essential for activating NF-κB. The IκB kinase (IKK) complex, which can be triggered by lipopolysaccharides (LPS), viral proteins, reactive oxygen species, cytokines, and other stimuli, phosphorylates Ser32 and Ser36 in IκBα's SRD region. This phosphorylation leads to IκBα's ubiquitination and subsequent degradation by the proteasome, freeing NF-κB to enter the nucleus and activate target genes. Mutations in IκBα that result in gain-of-function changes at Ser32/Ser36 can lead to an autosomal dominant form of anhidrotic ectodermal dysplasia with immunodeficiency.
    References

    技術サポート

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