Question

Your coworker purified a 20 kDa histidine-tagged protein using immobilized metal affinity chromatography (Qiagen Ni-NTA resin)....

Your coworker purified a 20 kDa histidine-tagged protein using immobilized metal affinity chromatography (Qiagen Ni-NTA resin). Her protein eluted partially in the wash step and also eluted with many contaminating proteins. How would you suggest trouble-shooting this experiment to improve the purification?

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Answer #1

The 20kDA histidine tagged protein was immobilized with help of metal affinity chromatography and while it was being eluted, it also eluted many contaminating proteins. In order to reduce the binding of the unspecific proteins to the resin, the buffer must contain imidazole and the resin must be limited. If the resin is saturated with the protein of interest, it does not let the contaminants to compete for a place to bind. The size of the column must be also reduced so that it does not pass on a lot of contaminants with it. Alternatively in order to break the bonds between the contaminants and the column, high salt concentration can be used

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