• 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

        Bacterial Expression of IRS-1 containing GST-fusion Proteins

        互联网

        781

        1.  Grow cells in 5ml (or more) of LB-Amp overnight for a starter culture.
         
        2.  Grow larger culture using the overnight culture as a seeding culture.  The culture is grown in LB-Amp medium.  Aerate well with shaking at 37 C until OD at 600nm is ~0.6.
         
        3.  Add the appropriate amount of IPTG stock solution to the culture to obtain a final IPTG concentration of ~2mM.
         
        4. Continue shaking for several hours up to overnight.
         
        5.  Spin down cells and resuspend pellet in 1/100 initial volume of culture into lysis buffer --  PBS-CMF, 2mM EDTA, 10mM DTT, PMSF, benzamidine and 0.5M NaCl.
         
        6.  Cells are lysed by sonication at 4 C.
         
        7.  Add Triton X-100 to 1% final concentration.
         
        8.  Centrifuge at 20 - 30 K rpm in ultracentrifuge at 4 C for 30 min.
         
        9.  Filter sample through a 0.45m pore size membrane.  Mix this filtered supernatant with glutathione Sepharose 4B which has been washed with PBS-CMF, 10mM DTT, 0.5M NaCl. 
         
        10.  Rotate the filtered supernatant/sepharose at 4 C for 30-60min.
         
        11.  Spin, remove liquid and wash the sepharose 2x with PBS-CMF, 10mMDTT, 0.5M NaCl.
         
        12.  Elute the fusion protein from the sepharose 3x with 10mM glutathione, 50mM Tris pH8.0, 10mM DTT, 0.5M NaCl.
         
        13.  Dialyze fusion protein against 50mM Tris pH8.0, 10mM DTT, 0.5M NaCl

        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