ホームページ 一次抗体 PFKM Rabbit mAb For research use only.
Not for use in humans.

PFKM Rabbit mAb

Catalog No.: F2565

    • Lane 1: Rat skeletal muscle
      Lane 2: Mouse skeletal muscle
      Lane 3: Ramos
      Lane 4: SHSY5Y
      Lane 5: HeLa
    1/
    サイズ （液体） 価格(税別) 在庫状況
    JPY 23500 国内在庫なし(納期7~10日)
    JPY 35500 国内在庫なし(納期7~10日)
    JPY 53500 お問い合わせ

    代表番号: 045-509-1970|電子メール：[email protected]
    よく尋ねられる質問

    キーポイント

    タンパク質の局在： 細胞質, 細胞質小胞, 小胞体, ゴルジ装置, リソソーム, 細胞内膜系, ミクロソーム, ミトコンドリア, ミトコンドリア外膜, 细胞核。
    WB
    RIPA/Tris-HCL Lysis Buffer バッファーでのライセート調製を推奨します。

    使用情報

    Dilution
    1:1000
    1:300
    1:100 - 1:500
    Application
    WB, IF, FCM
    Source
    Rabbit
    Reactivity
    Human, Mouse, Rat, Pig
    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
    85 kDa
    ポジティブコントロール Human fetal brain; Human fetal heart; Mouse skeletal muscle; Rat skeletal muscle; Pig heart; Pig skeletal muscle; HeLa; SH-SY5Y; Ramos
    ネガティブコントロール

    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/Tris-HCL 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/Tris-HCL 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/Tris-HCL 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:
     
    Sample Preparation
    1. Adherent Cells: Place a clean, sterile coverslip in a culture dish. Once the cells grow to near confluence as a monolayer, remove the coverslip for further use.
    2. Suspension Cells: Seed the cells onto a clean, sterile slide coated with poly-L-lysine.
    3. Frozen Sections: Allow the slide to thaw at room temperature. Wash it with pure water or PBS for 2 times, 3 minutes each time.
    4. Paraffin Sections: Deparaffinization and rehydration. Wash the slide with pure water or PBS for 3 times, 3 minutes each time. Then perform antigen retrieval.
     
    Fixation
    1. Fix the cell coverslips/spots or tissue sections at room temperature using a fixative such as 4% paraformaldehyde (4% PFA) for 10-15 minutes.
    2. Wash the sample with PBS for 3 times, 3 minutes each time.
     
    Permeabilization
    1.Add a detergent such as 0.1–0.3% Triton X-100 to the sample and incubate at room temperature for 10–20 minutes.
    (Note: This step is only required for intracellular antigens. For antigens expressed on the cell membrane, this step is unnecessary.)
    Wash the sample with PBS for 3 times, 3 minutes each time.
     
    Blocking
    Add blocking solution and incubate at room temperature for at least 1 hour. (Common blocking solutions include: serum from the same source as the secondary antibody, BSA, or goat serum.)
    Note: Ensure the sample remains moist during and after the blocking step to prevent drying, which can lead to high background.
     
    Immunofluorescence Staining (Day 1)
    1. Remove the blocking solution and add the diluted primary antibody.
    2. Incubate the sample in a humidified chamber at 4°C overnight.
     
    Immunofluorescence Staining (Day 2)
    1. Remove the primary antibody and wash with PBST for 3 times, 5 minutes each time.
    2. Add the diluted fluorescent secondary antibody and incubate in the dark at 4°C for 1–2 hours.
    3. Remove the secondary antibody and wash with PBST for 3 times, 5 minutes each time.
    4. Add diluted DAPI and incubate at room temperature in the dark for 5–10 minutes.
    5. Wash with PBST for 3 times, 5 minutes each time.
     
    Mounting
    1. Mount the sample with an anti-fade mounting medium.
    2. Allow the slide to dry at room temperature overnight in the dark.
    3. Store the slide in a slide storage box at 4°C, protected from light.

    Datasheet & SDS

    生物学的記述

    Specificity

    PFKM Rabbit mAb detects endogenous levels of total PFKM protein.
    Synonym(s)
    PFK-1,PFKM
    Uniprot ID
    P08237
    Clone
    F13D16
    Background

    Phosphofructokinase muscle isoform (PFKM) is a critical regulatory enzyme in glycolysis, catalyzing the ATP-dependent phosphorylation of fructose-6-phosphate to fructose-1,6-bisphosphate, a key rate-limiting step. Predominantly expressed in muscle and neuronal tissues, PFKM forms homotetramers in muscle and heterotetramers with other isoforms like PFKL in non-muscle tissues, enabling tissue-specific regulation. Structurally, PFKM (~85 kDa) features an N-terminal catalytic region, a C-terminal region with allosteric sites for ligands (e.g., ATP, citrate), and a tetrameric assembly that allows cooperative enzymatic activity. It interacts with NOS1 via the PDZ domain, enabling rapid glycolytic regulation and apoptosis resistance in neurons through S-nitrosylation at Cys351, which stabilizes the tetramer and reduces feedback inhibition. Dysregulation of PFKM-NOS1 interactions is implicated in muscle ischemia, damage, and cancer progression, with elevated PFKM and NOS1 levels correlating with advanced ovarian cancer stages.
    References

    技術サポート

    ストックの作り方、阻害剤の保管方法、細胞実験や動物実験の際に注意すべき点など、製品を取扱う時に問い合わせが多かった質問に対しては取扱説明書でお答えしています。

    Handling Instructions

    他に質問がある場合は、お気軽にお問い合わせください。

    * 必須

    大学・企業名を記入してください
    名前を記入してください
    電子メール・アドレスを記入してください 有効なメールアドレスを入力してください
    お問い合わせ内容をご入力ください