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A continuous‐flow isoelectric focusing system for protein fractionation: Its description and application in the partial purification of choline acetyltransferase
Author(s) -
Basset Paul,
Froissart Chantal,
Vincendon Guy,
Massarelli Raffaele
Publication year - 1980
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.1150010309
Subject(s) - isoelectric focusing , chromatography , fractionation , chemistry , hemoglobin , continuous flow , biochemistry , enzyme , physics , mechanics
Abstract A continuous‐flow isoelectric focusing (IEF) system for protein fractionation, using a non‐commercial separation chamber and carrier ampholytes synthesized in the laboratory, is described. The resolving power and the sample‐loading capacity have been tested by the fractionation of hemoglobin mixtures. Hb A 2 and HbA (ΔpI 0.4) were separated with a sample loading of 4 g of hemoglobin per day. The maximal temperature in the gel was 5 °C, the average field strength 30 V/cm and the residence time 6 h. The continuous‐flow IEF system was further used as a preliminary step in the purification of choline acetyltransferase (EC. 3.2.1.6) from human placenta. The results obtained with choline acetyltransferase indicated that continuous‐flow IEF can be used for the purification of enzymes when other preparative IEF systems are not effective, particularly when the sample is present in large volume.