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Column isoelectric focusing in natural pH gradients generated by biological buffers
Author(s) -
Vaidya Narendra R.,
Gothoskar Balwant P.,
Banerji Ashutosh P.
Publication year - 1990
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150110210
Subject(s) - isoelectric focusing , chromatography , immobilized ph gradient , chemistry , isoelectric point , electrophoresis , lysozyme , bovine serum albumin , biochemistry , enzyme
Using 2 or 3 simple Good z witterionic 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, respecitively, 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 sulfategel 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.