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【求助】镍柱再生后结合效率很低

字体: 小 中 大 | 打印 发表于: 2013-6-08 13:51    作者: qianqin1977    来源: 分析测试百科网

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【求助】镍柱再生后结合效率很低
请教各位大侠
我用镍柱纯化可溶性蛋白，过了几次柱子之后，凝胶珠就泛白了，我想继续纯化，所以就打算再生镍柱
我用的方法是，用2倍柱体积的0.1M NaOH 过柱子目的是洗脱凝胶珠上的所有蛋白，然后再用6-7倍柱体积的水洗，然后再加入0.1M NiSO4 目的是让凝胶珠重新挂上镍。
但是再次纯化蛋白后发现目的蛋白大部分没有结合上，用wash buffer 洗脱后，绝大部分都洗脱下来，而在elution的步骤中目的蛋白了非常少。
不知道我的步骤中有什么不妥之处，望各位大虾指教！
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【求助】镍柱再生后结合效率很低

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  • memory (2013-6-08 13:52:49)

    相关疾病：
    阿尔茨海默病
    first remove residual Ni2+,
    wash with 5 column volumes 20 mM sodium phosphate, 0.5 M
    NaCl, 50 mM EDTA, pH 7.4. Remove residual EDTA by
    washing with at least 5 column volumes of binding buffer
    followed by 5 column volumes of distilled water before
    recharging the column.To recharge the water-washed column, load 0.5 column volumes
    of 0.1 M NiSO4 in distilled water. Salts of other metals, chlorides
    or sulfates, may also be used (see Optimization).
    Wash with 5 column volumes of distilled water followed by 5
    column volumes of binding buffer (to adjust pH) before storage
    in 20% ethanol.
    In some applications, substances such as denatured proteins or
    lipids cannot be eluted in the regeneration. These can be
    removed by Cleaning-in-Place.

  • bamboo16 (2013-6-08 13:53:39)


    Ni柱的名称？最好按照说明书进行再生使用。

  • qianqin1977 (2013-6-08 13:54:03)


    镍柱是QIAGEN公司的填料。说明书上没有给出再生的步骤和说明。
    我还想请问一下有关QIAGEN的镍柱凝胶珠填料，1ml的凝胶珠能结合多少蛋白？谢谢！

  • 49888 (2013-6-08 13:54:48)


    主要是看一下镍柱的再生

  • yonger (2013-6-08 13:56:15)


    相关疾病：
    阿尔茨海默病

    =====================

    主要是看一下镍柱的再生

    ===========================================================================================================

    Regeneration by stripping and recharging
    Stripping and recharging of Ni-NTA column is usually not necessary. If an increase in back pressure or significant contamination of the resin is observed, a cleaning-in-place procedure usually restores performance. However, if performance is still not satisfactory, the Ni-NTA resin in the cartridge can be stripped and recharged using the protocol below.

    1. Strip the resin by washing with 10 resin volumes of stripping buffer (50 mM Na phosphate; 300 mM NaCl; 100 mM EDTA;pH 8.0).
    2. Wash the resin with 20 resin volumes of deionized water.
    3. Recharge the water-washed column by loading 2 resin volumes of 100 mM NiSO4 (in deionized water).
    4. Wash with 10 resin volumes of deionized water and re-equilibrate with 10 resin volumes of 1xPBS. The column is now ready for use. Store column in 20–30% ethanol or 10–100 mM NaOH
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【求助】镍柱再生后结合效率很低