Advantages of the fixed-angle rotor for the separation of density-labelled from unlabelled proteins by isopycnic equilibrium centrifugation.
A comparison has been made between the swinging-buckets rotor and the fixed-angle rotor for density-labelling experiments. The enzymes examined were beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), L-phenylalanine ammonia-lyase (EC 4.3.1.5) and acid phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.2); it was found that the fixed-angle rotor offered greater separation and resolution and shorter equilibration times compared with the swinging-buckets rotor at the same angular velocity. By exploiting the greater flexibility offered by the fixed-angle rotor it is possible to obtain either a 6-fold improvement in separation or a 6-fold reduction in equilibration time. The significance of these results for density-labelling work is discussed.
Johnson
C
C
University of Nottingham School of Agriculture, Sutton Bonington, Loughborough, Great Britain.
Attridge
T
T
Smith
H
H
D000085:Acetates / Q000737:chemistry; D000135:Acid Phosphatase / Q000737:chemistry; D002021:Buffers; D002499:Centrifugation, Density Gradient; D002500:Centrifugation, Isopycnic / Q000379:methods*; D018553:Cucumis sativus / Q000201:enzymology; D004867:Equipment Design; D006863:Hydrogen-Ion Concentration; D015233:Models, Statistical; D018532:Pisum sativum / Q000201:enzymology; D010650:Phenylalanine Ammonia-Lyase / Q000737:chemistry; D011506:Proteins / Q000032:analysis / Q000302:isolation & purification*; D013696:Temperature; D001616:beta-Galactosidase / Q000378:metabolism
D000085:Acetates; D002021:Buffers; D011506:Proteins; D000135:Acid Phosphatase; D001616:beta-Galactosidase; D010650:Phenylalanine Ammonia-Lyase