• 我要登录|
  • 免费注册
    |
  • 我的丁香通
    • 企业机构:
    • 成为企业机构
    • 个人用户:
    • 个人中心
  • 移动端
    移动端
丁香通 logo丁香实验_LOGO
搜实验

    大家都在搜

      大家都在搜

        0 人通过求购买到了急需的产品
        免费发布求购
        发布求购
        点赞
        收藏
        wx-share
        分享

        Immunoprecipitation

        互联网

        1672

        Hancock Laboratory Methods.Department of Microbiology and Immunology,

        University of British Columbia,British Columbia,Canada

        http://www.cmdr.ubc.ca/bobh/showmethod.php?methodid=28

        MATERIALS:

        Running buffer:

        2 ul 1.25M Tris-HCl, pH 6.
        35 ul distilled water
        2.5 ul 2-mercaptoethanol
        12.5 ul 10% SDS
        10 ul 80% glycerol
        2 ul bromphenol blue
        TNE buffer:

        10 mM Tris-HCL,pH 8.0

        10 mM NaCl

        0.5 mM EDTA

        METHODS:

        Resuspend cell pellet in buffer TNE containing 1% NP40 detergent and vortex.

        Incubate for 30 minutes on ice or at 37℃ for 10 to 15 minutes.

        Pellet cell debris in an Eppendorf mini centrifuge (10,000 x g)for 3 minutes.

        Decant the supernatant into a fresh tube and add 40-50 μl of antiserum.

        Incubate on ice for 2 hours or overnight at 4℃.

        Add 100 μl of S.aureus protein A and 100 μl  5% BSA in TNE.

        Incubate on ice for 2 hours.

        Pellet the immune complexes and wash twice with 1ml 1%NP40,0.5% Na deoxychloate,0.1% SDS in TNE and sonicate once to resuspend.

        Resuspend the final pellet in 70 μl running buffer.

        Boil in a water bath for 90 seconds and centrifuge for 1 minute to remove S.aureus (saving supernatant).

        Refrigerate,if the preparation is to be stored before use (e.g.SDS-PAGE).

        ad image
        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