• 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

        PIC Cross-linking and Immune Precipitation

        互联网

        1838

        PIC Cross-linking and Immune Precipitation

        James Fishburn,Hahn Lab March 2006

        References

        Fishburn et.al.,2005,Molecular Cell,vol.18 #3: Experimental Procedures pg.376

        Immobilized Template assay (Hahn lab website)

        Notes

        DTT must be omitted from all buffers

        1 M KOAc seems to be the upper limit for solubility of NP40.If a precipitate forms,lower the KOAc concentration slightly.

        A 'no UV' control is not required for this procedure (see step 7).

        Procedure

        1.Prepare templates per standard protocol

        2.Dialyze extracts using 0.02 micron discs on buffer C + 75 mM ammonium sulfate + protease inhibitors for 1 hr at 4 deg to remove DTT (100-150 microliter extract per 10ml buffer C)

        3.Add PEAS labeled activator to templates and bind 10' at RT

        4.Make reaction mix and form 2X PICs (200 microliter)per standard protocol

        5.Wash PICs 3 x 400 microliter txn wash buffer

        6.Resuspend PICs in 200 microliter txn wash buffer

        7.Do not split reactions into 100 microliter aliquots as is standard for cross-linking reactions

        8.Cross-link samples in the UV oven: energy = 1250 units with 365nm bulbs

        9.Concentrate beads,discard supernatant

        10.Resuspend in 20 microliter txn wash buffer + 2 microliter 1 M DTT

        11.Incubate 10' at RT,mix occasionally

        12.Concentrate beads,discard supernatant

        13.Resuspend beads in 100 microliter disruption buffer (txn wash buffer + 1 M KOAc final concentration)

        14.Incubate 30' at RT with gentle mixing (Dynal mixer)to disrupt PICs

        15.Concentrate beads,transfer supernatant to new tube containing 10 microliter M2-±Flag Sepharose

        16.Incubate IP reactions 1 hr at RT with mixing on Dynal mixer

        17.Collect Sepharose resin by centrifugation,discard supernatant

        18.Wash resin 1 x 100 microliter disruption buffer

        19.Wash resin 1 x 100 microliter txn wash buffer to reduce salt ubiquitous adsf

        20.Elute bound proteins with 15 microliter 1X SDS sample buffer + 1.5 microliter 1 M DTT: incubate at 70deg for 10' with mixing in Thermomixer

        21.Transfer eluates to new tubes,store at -20 deg

        Analysis

        Analyze by Phosphorimager and Western

        1.Run samples on 4-12% Bis-Tris or other NuPAGE

        2.Transfer to Immobilon FL membrane

        3.Air dry membrane

        4.Expose to Phosphorimager screen for 2-7 days

        5.Rewet membrane in blocking buffer-PBS

        6.Perform Western for ±Flag and/or other proteins of interest per standard protocol

        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