Hi,
I purify protein by IMAC purification by increasing imidazole concentration. However, in the chromatograph sometimes it is difficult to see a clear peak from A280 of protein beacuse of the higher signal from imidazole.
Do you know if some company sell a specific imidazole with a lower signal at 280nm? Or do you have other suggestion to solve this problem?
Thanks a lot!
No chance, I'm afraid. Absorption at 280 is an intrinsic property of the reagent. You could try to eluate with EDTA instead. However, to regenerate the column, you need to flush it with water and then recharge it with Nickel chloride or sulphate. Then flush again with water, before equilibrating with PBS. (The phosphate from PBS will precipitate free nickel ions otherwise and destroy your column).
Actually, pure imidazole has very weak absorbance at 280 nm. The absorbance seen in solutions of imidazole is due to contaminants. Different imidazole products have different levels of UV absorbance at 280 nm. I faced the same problem and switched to a more expensive, higher-purity imidazole and nearly eliminated the background absorbance. Unfortunately, the low-absorbance products tend to be relatively expensive because of their high purity . I suggest you ask for samples from the chemical suppliers you use, make a 1 M solution of each, and measure their absorbances with a spectrophotometer. Either that, or buy the highest purity grade you can afford.
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