Q：柱子反压过高是什么原因？

A：填料可能被堵塞：裂解液中可能含微小的固体颗粒，建议上柱前使用滤膜（0.22µm 或 0.45µm）过滤，或离心去除。

Q：为什么洗脱组分中无目的蛋白？

A：蛋白可能是包涵体：可通过电泳检测裂解液，分析上清中是否有目的蛋白，包涵体蛋白需按照包涵体蛋白的纯化方式；

Q：洗脱组分不纯（含多种蛋白）是什么原因？

A： 样品中含有其他 His 标签蛋白：通过调节 pH 值或咪唑浓度来优化洗杂条件。再使用其他纯化手段（如去离子交换，疏水等）进一步纯化洗脱组分。

Q：为什么填料颜色会变浅或变成白色？

A：镍离子脱落或者剥离，按照填料再生的操作重新挂镍离子。

Q：产品规格的体积指的是什么？

A：指的是填料的体积，不包括保护液的。

Q：填料流速慢的原因？

A：1.柱子在清洗平衡步骤流速减慢，柱子下筛板堵塞或者是试剂中有杂质堵塞。2.上样过程中流速逐渐降低，样品中有不溶物 堵塞填料或者是样品粘稠。少数情况蛋白挂柱后 流速也会减慢。 3.洗杂洗脱步骤流速减慢，蛋白不稳定 在柱子上沉淀，可以选择低温条件纯化，或者是在整个纯化过程中样品试剂中加入甘油保护蛋白。

Q：说明书步骤的5-10倍柱体积具体指的的是什么？

A：指的是5-10倍的填料体积。

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