Performance evaluation of a focusing buffer developed for chromatofocusing on high-performance anion-exchange columns

Abstract

We have developed a single focusing buffer for generation of wide-range pH gradients during chromatofocusing on high-performance anion-exchange columns. Various properties of this focusing buffer have been compared to those of commercially available polyampholyte buffers as well as non-polyampholyte buffers. Relative to the other buffers evaluated, our focusing buffer exhibited lower UV absorbance and only slightly higher conductivity at effective operating concentrations. No other buffer tested was able to produce linear pH gradients (from pH 8.1 to 4) of a quality better than that obtained using our focusing buffer. Using our focusing buffer with 25-cm Bakerbond PEI columns, several purified proteins (10 mg each) were eluted at pH values near to or identical with their elution pH or p I values as reported elsewhere. High-performance chromatofocusing with this same system revealed that the surface charge distribution of estrogen receptor proteins (50–100 fmol/mg cytosol protein) in calf uterine cytosol was similar to that reported by us previously. Since our focusing buffer is composed of low-molecular-weight components, in contrast to polyampholytes, these components are more easily separated from proteins and do not interfere with protein stains or dyes. These results confirm and extend the general utility of simple focusing buffer systems as alternatives to the larger polyampholytes for chromatofocusing.