For LDHA enzyme purification using Ni2+-NTA chromatography, the following buffers were used: (1) Ni binding buffer with 20mM imidazole (2)Ni washing buffer with 100mM imidazole. However, impurities were still found, how can i improve my wash step?
Should I increase the concentration of imidazole?
Is it a native or recombinant protein? Anyway, an imidazole gradient will help you resolve the adsorbed proteins.
Do you add NaCl in the binding buffer? You can try from 200 mM to 1M. Also a higher imidazole (from 25 mM to 50 mM) concentration in the binding buffer will prevent unwanted binding...Then, when you elute with 100 mM, you may have a better profile.
Sorry for late reply.
It is a recombinant protein. 500mM of NaCl is added in the binding buffer.
But will increased imidazole concentration in binding buffer causes the increased competences between 6His-tag on the N-terminal of protein and the imidazole, resulting in the low yield of target protein?
How does NaCl help in the purification step?
Higher imidazole concentration in the binding buffer should prevent unspecific fixation of unwanted protein (the competition for Ni NTA favours the anchoring of the 6His tag-targeted protein) and at the end you elute a cleaner product.
Higher imidazole concentration in the binding buffer should prevent unspecific fixation of unwanted protein (the competition for Ni NTA favours the anchoring of the 6His tag-targeted protein) and at the end you elute a cleaner product. - Veronique Piller
Thanks for your help and suggestions!!
Is it a native or recombinant protein? Anyway, an imidazole gradient will help you resolve the adsorbed proteins. - Omar Gonzalez-Ortega.
Thanks for everyone who replied. I will try to explain it in my lab report.:)
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