丁香实验_LOGO
登录
搜索
    搜索
    提问
    我要登录
    ｜免费注册
    点赞
    收藏
    wx-share
    分享

    Nuclear enrichment

    互联网

    1074

    实验试剂

     

    1. Buffer A

        10mM HEPES

        1.5mM MgCl2

        10mM KCl

        0.5mM DTT

        0.05% NP40 (or 0.05% Igepal or Tergitol)

        pH 7.9

    2. To prepare 250ml stock of Buffer A:

        HEPES: 1M = 238.3g/litre, therefore 10mM = 0.59g/250ml

        MgCl2 : 1M = 203.3g/litre, therefore 1.5mM = 0.076g/250ml

        KCl: 1M = 74.5g/litre, therefore 10mM = 0.187g/250ml

        DTT: 1M = 154.2g/litre, therefore 0.5mM = 0.019g/250ml

        NP40 = 0.05% (v/v)

    3. Buffer C

        5mM HEPES

        1.5mM MgCl2

        0.2mM EDTA

        0.5mM DTT

        26% glycerol (v/v)

        pH 7.9

    4. To prepare 250ml stock of Buffer C:

        HEPES: 1M = 238.3g/litre, therefore 5mM = 0.295g/250ml

        MgCl2 : 1M = 203.3g/litre, therefore 1.5mM = 0.076g/250ml

        EDTA: 1M = 372.2g/litre, therefore 0.2mM = 0.0186g/250ml

        DTT: 1M = 154.2g/litre, therefore 0.5mM = 0.019g/250ml

        26% Glycerol (v/v) = 65ml

        4.6M NaCl

        87.66g/326ml H2 0

    实验步骤

     

    1. Requires large 12ml petri dishes. Prepare 1ml of Buffer A with added cocktail of protease inhibitors from a frozen stock and store on ice.

    2. Add 500µl of Buffer A per large petri dish on ice and scrape thoroughly, leave on ice for 10 min (NP40 primarily lyses plasma membrane and leaves other membranes more intact than Triton X-100 does).

    3. Centrifuge at 4o C at 3000 rpm for 10 min.

    4. Remove supernatant and keep (this will contain everything except large plasma membrane pieces, DNA, nucleoli), extract out 10µl for Bradford assay.

    5. On ice, resuspend pellet in 374µl of Buffer C and add 26µl of 4.6M NaCl to give 300mM NaCl (high salt helps lyse membranes and forces DNA into solution).

    6. Homogenise with 20 full strokes in Dounce or glass homogeniser on ice.

    7. Leave on ice for 30 min

    8. Centrifuge at 24,000g for 20 min at 4o C

    9. Aliquot supernatant, remove 10µl for Bradford assay and store at –70o C.

    相关产品推荐

    Proliferating Cell Nuclear Antigen (PCNA)，阿拉丁

    Proliferating Cell Nuclear Antigen (PCNA)，阿拉丁

    ￥1400.90

    Small Nuclear Ribonucleoprotein Sm D (Sm D1, D2, D3) 抗体, 生物素偶联

    Small Nuclear Ribonucleoprotein Sm D (Sm D1, D2, D3) 抗体, 生物素偶联

    ￥11025

    Small Nuclear Ribonucleoprotein Sm D (Sm D1, D2, D3) 抗体, RPE偶联

    Small Nuclear Ribonucleoprotein Sm D (Sm D1, D2, D3) 抗体, RPE偶联

    ￥12950

    Small Nuclear Ribonucleoprotein Sm D (Sm D1, D2, D3) 抗体, PerCP偶联

    Small Nuclear Ribonucleoprotein Sm D (Sm D1, D2, D3) 抗体, PerCP偶联

    ￥14350

    Small Nuclear Ribonucleoprotein Sm D (Sm D1, D2, D3) 抗体, FITC偶联

    Small Nuclear Ribonucleoprotein Sm D (Sm D1, D2, D3) 抗体, FITC偶联

    ￥12950

    相关方法

    设 计 和 克 隆 一 个 sh R N A 到 慢 病 毒 沉 默 载 体 中 ：版 本 A

    设 计 和 克 隆 一 个 s h R N A 到 慢 病 毒 沉 默 载 体 中 ：版 本 B

    使 用 其 他 佐 剂 制 备 多 克 隆 抗 体 的 免 疫 接 种 法

    推荐阅读

    核膜 nuclear membrane，nuclear envelope

    Human DC Enrichment Kit

    Visualizing Gene-Set Enrichment Results Using the Cytoscape Plug-in Enrichment Map

    提问
    扫一扫
    丁香实验小程序二维码
    实验小助手
    丁香实验公众号二维码
    扫码领资料
    反馈
    TOP
    打开小程序