Column isoelectric focusing in natural pH gradients generated by biological buffers

Using 2 or 3 simple Good zwitterionic buffers at a 16 or 18 mmol/L final column concentration of the mixture, natural pH gradients of 4 to 8 and 3 to 9.5, respectively, were generated in a liquid LKB column. The pH gradients, stabilized by an anticonvective sucrose gradient, were linear, reproducible and stable in the electric field up to 5h. The pH gradients were used for isoelectric focusing of a number of impure proteins such as human hemoglobin, bovine serum albumin and chicken egg white lysozyme. The protein components could be well separated in the gradient, were easily recovered and appeared to be quite pure when analyzed by sodium dodecyl sulfate-gel electrophoresis. Furthermore, the pH gradient 4-8 was effectively used to isolate one of the acidic isozyme (pI 5.6) components of mouse liver alcohol dehydrogenase (EC 1.1.1.1) in an enzymatically active state, suggesting that the procedure does not denature proteins. The low cost, the ease with which the pH gradients are formed, their linearity, stability for a sufficient period to allow proteins to reach equilibrium and their subsequent recovery from buffer eluates should make the procedure interesting for electrofocusing of proteins.