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Imidazole has traditionally been added to the wash buffer to modulate non-specific binding when purifying HIS-tagged proteins with affinity gels. However, the HIS-Select® technology is so selective that it requires much less imidazole (0–10 mM) in the wash buffer than has traditionally been used (Figure 1). Even with no imidazole in the wash buffer, note how little non-specific binding occurs when using the HIS-Select Affinity Gel in lane 2 in comparison to the lanes 3–5. Furthermore, washing with the low concentration of 10 mM imidazole, non-specific binding has been virtually eliminated (lane 6) with the HIS-Select Affinity Gel. Using concentrations of imidazole higher than 10 mM will not further decrease non-specific binding.
Figure 1:
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Lysates from E. coli containing 8 mg histidine-tagged protein per ml resin were loaded onto the resins in the presence of either 0 or 10 mM imidazole. These were incubated for 30 min at room temperature while rotating. Resins were washed three times with wash buffer (50 mM NaPO4, 300 mM NaCl, pH 8) and HIS-tagged protein was eluted with 250 mM imidazole. Eluted protein was run on a gel and stained with EZBlue stain (G1041). |
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