问题一：目的蛋白结合效率低或不结合
可能原因：蛋白序列出现移码等错误
解决办法：测序确认，调整阅读框以获得GST.Tag融合表达蛋白；选择适宜的宿主菌，如蛋白酶缺陷型大肠杆菌，稀有密码子宿主菌Rosetta系列。
可能原因：GST 融合蛋白被机械裂解的方法变性（比如超声）
解决办法：过度超声产热会破坏标签蛋白，建议采用超高压破碎。
可能原因：GST 融合蛋白发生聚集沉淀
解决办法：在裂解buffer和其他缓冲体系中加入DTT，经验告知，1-20mM的DTT 会显著增加某些GST融合蛋白的结合。
可能原因：GST融合蛋白被过度稀释
解决办法：重新浓缩样品，增加蛋白浓度。
可能原因：融合蛋白可能改变了GST 的构象，因此降低了GST 标签蛋白的结合能力。
解决办法：检测所使用的pGEX 载体中GST的结合。准备带有所使用的pGEX的细胞超声裂解物，检测其与层析柱的结合。如果结合的很好，则可能是标签蛋白改变了GST 的构象，因此降低了GST 标签蛋白的亲和力。可以通过降低结合的温度到4°C限制洗涤来改善结果。
可能原因：结合缓冲条件不适宜
解决办法：低于pH6.5或高于pH8时，融合蛋白与层析柱结合不充分导致结合效率低，请确认磁珠在用于目的蛋白纯化前经过pH6.5~8.0缓冲液充分的平衡。
可能原因：层析柱使用次数过多，杂质干扰
解决办法：层析柱再生或使用新的层析柱。
问题二：目的蛋白洗脱不下来或洗脱率低
可能原因：洗脱缓冲液的体积太少
解决办法：增加洗脱缓冲液的体积。
可能原因：洗脱的时间不够
解决办法：增加洗脱缓冲液与磁珠反应的时间。
可能原因：洗脱缓冲液中谷胱甘肽浓度太低
解决办法：增加洗脱缓冲液中谷胱甘肽的浓度
可能原因：洗脱缓冲液的pH过低
解决办法：调节洗脱缓冲液pH值至8-9 。
可能原因：洗脱缓冲液的离子强度过低
解决办法：增加洗脱缓冲液中的离子强度，在洗脱缓冲液中加入0.1~0.2M的氯化钠也会改善洗脱效果。
可能原因：洗脱缓冲液中谷胱甘肽失效被氧化
解决办法：洗脱缓冲液现配现用。
问题三：洗脱的蛋白中有杂质
可能原因：GST融合蛋白被蛋白酶部分降解
解决办法：加入蛋白酶抑制剂PMSF。注：丝氨酸蛋白酶抑制剂必须在使用凝血酶或凝血因子Xa 前除去。
可能原因：在宿主细菌中蛋白被降解
解决办法：使用蛋白酶缺陷型菌株lon-或ompT，或ompT和lon双缺陷型菌株。
可能原因：细胞破碎时超声处理过度
解决办法：降低裂解时间，机械裂解前加入溶菌酶（0.1 倍体积的10 毫克/毫升溶菌酶溶液，溶菌酶保存在25mM Tris-HCl,pH8.0 ）可能会使结果变好。避免发泡，因为这可能使标签蛋白变性。过分裂解也会导致宿主细胞蛋白和GST 标签蛋白共纯化。
可能原因：分子伴侣可能被共纯化了
解决办法：多余的条带可能由于共纯化一些分子伴侣所造成。这些分子伴侣参与大肠杆菌中新生成的蛋白的正确折叠。如：DnaK（分子量70 000）、DnaJ（分子量37 000）、GrpE（分子量40 000）GroEL （分子量57 000）、GroES（分子量10 000 ）。一些从这些共纯化的蛋白中分离GST融合蛋白的方法已经发表。
问题四：目的蛋白酶切后电泳检测发现多条条带
可能原因：蛋白酶切发生在宿主细菌内
解决办法：检测条带何时出现：如果确定多余的条带在Precission Protease，凝血酶，凝血因子Xa切割前不存在，这些条带可能是在宿主细菌中被降解的结果。目的蛋白可能含有Precission Protease，凝血酶，凝血因子Xa 的切割位点，请检查序列。
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