Should a pH- or imidazole-based elution be used?
Should a pH- or imidazole-based elution be used?
This is the researchers' choice. We recommend using imidazole since it minimizes nonspecific binding, but imidazole impurities and his-tagged proteins both absorb at 280 nm, so elution peaks can be difficult to distinguish. However, Takara Bio's buffers are made with low-absorbance imidazole, so the only molecule absorbing at 280 nm is the his-tagged protein. The choice may also depend on downstream applications. A pH-based elution will work well as long as the protein of interest is stable at the lower pH, usually around pH 5.0 when using a 50 mM sodium acetate/300 mM NaCl buffer to elute. For the best pH elution results, use a pH gradient.