Production of dye-binding esterase reactor after separation and detection using combined native isoelectric focusing and blue native electrophoresis

Abstract

When cytosolic proteins from mouse liver were separated by a combined method of native isoelectric focusing (IEF) and blue native electrophoresis, spots at pI 6.7/180,000 Da, pI 6.5/100,000 Da and pI 6.4/70,000 Da were detected by Coomassie Brilliant Blue. After separation and detection, the native enzymes were extracted by native electrophoresis and immobilized on the reverse-phase chromatography media ZipTip to produce an enzyme reactor. The hydrolysis activity of 4-methylumebelliferyl acetate by the spots at pI 6.7/180,000 Da and pI 6.4/70,000 Da was 3.0 and 2.4 times, respectively, greater than that with no enzyme. The method can be applied to systematically produce biological reactors after separation and detection of enzymes by the combined method of native IEF and blue native electrophoresis.