When using Ni-NTA affinity chromatography gel beads (Cat.No.: HY-K0210, bought from MCE) for His tag protein purification, the color of the gel beads was observed to change (for example, turning green, yellow, or other hue changes) after incubation with cell lysate.
Is this normal?
Possible influencing factors include: whether the reducing agent (such as DTT) in the lysate causes Ni2+ to fall off, non-specific interactions between cellular components (such as pigments or metal binding proteins) and gel beads, or whether the incubation conditions (pH/temperature) affect the stability of metal ions.
- Has anyone encountered similar situations in similar experiments?
- Looking forward to sharing experiences or suggestions~
[Troubleshooting Questions are selected from MCE customer consultation emails.]
Analysis and suggestions on the color change of Ni-NTA affinity chromatography medium (Cat.No.: HY-K0210):
1. Lighter color:
Possible cause: Ni2+ loss from the medium surface.
Suggested measures: Optimize the binding buffer components (such as adjusting pH, imidazole concentration, etc.), and regenerate the medium (such as re-coupling Ni2+ after treatment with eluent/regeneration solution according to the instructions).
2. The color turns brown:
Possible cause: The buffer contains strong reducing agents such as DTT, which causes Ni²⁺ to be reduced or the coordination structure to be destroyed.
Suggested measures: Reduce the DTT (Cat.No.: HY-15917) concentration to below 1 mM, or replace it with β-mercaptoethanol (≤50 mM) as a reducing agent.
The answer to this question comes from MCE Technical Support.
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