Rate zonal sedimentation of proteins in one hour or less.
Rate zonal sedimentation gives information about the shape and size of proteins, and is useful for investigating protein-protein interactions. However, rate zonal sedimentation experiments typically last approximately 1 day. In contrast, this report describes a rate zonal sedimentation method requiring 1 h or less. This was accomplished by centrifuging small density gradients (200 microliters) prepared with sucrose or OptiPrep in a fixed-angle rotor at high relative centrifugal force. By using small gradient volumes, the sample dilution that occurs with larger gradients and with many chromatographic techniques was also avoided. For a variety of proteins, plots of S20,w versus distance sedimented during centrifugation in a TLA 120.2 rotor were linear. As a practical application, sedimentation of the heterotrimeric stimulatory G protein and its dissociated alpha-subunit were determined. The results were similar to those obtained with 17- to 22-h centrifugations in an SW 50.1 rotor and agreed with previously published values. Long periods of centrifugation might preclude the study of some unstable proteins or the investigation of protein-protein interactions whose affinities are to low to survive the lengthy centrifugations required to carry out traditional rate zonal sedimentation experiments. A rate zonal sedimentation technique that rivals many chromatographic methods in celerity will help to circumvent these problems.
Basi
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NS
Membrane Biochemistry Section, NINDS, NIH, Bethesda, Maryland 20892, USA.
Rebois
R V
RV
D000818:Animals; D002417:Cattle; D002501:Centrifugation, Zonal / Q000295:instrumentation / Q000379:methods*; D005069:Evaluation Studies as Topic; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification; D011487:Protein Conformation; D011506:Proteins / Q000302:isolation & purification*; D013395:Sucrose; D013997:Time Factors
D011506:Proteins; D013395:Sucrose; D019204:GTP-Binding Proteins