title: Improving the thermal, radial, and temporal accuracy of the analytical ultracentrifuge through external references.
abstract: Sedimentation velocity (SV) is a method based on first principles that provides a precise hydrodynamic characterization of macromolecules in solution. Due to recent improvements in data analysis, the accuracy of experimental SV data emerges as a limiting factor in its interpretation. Our goal was to unravel the sources of experimental error and develop improved calibration procedures. We implemented the use of a Thermochron iButton temperature logger to directly measure the temperature of a spinning rotor and detected deviations that can translate into an error of as much as 10% in the sedimentation coefficient. We further designed a precision mask with equidistant markers to correct for instrumental errors in the radial calibration that were observed to span a range of 8.6%. The need for an independent time calibration emerged with use of the current data acquisition software (Zhao et al., Anal. Biochem., 437 (2013) 104-108), and we now show that smaller but significant time errors of up to 2% also occur with earlier versions. After application of these calibration corrections, the sedimentation coefficients obtained from 11 instruments displayed a significantly reduced standard deviation of approximately 0.7%. This study demonstrates the need for external calibration procedures and regular control experiments with a sedimentation coefficient standard.
authors.affiliation: Laboratory of Molecular Biology, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Rodolfo
authors.initials: R
authors.lastname: Ghirlando
authors.forename: Andrea
authors.initials: A
authors.lastname: Balbo
authors.forename: Grzegorz
authors.initials: G
authors.lastname: Piszczek
authors.forename: Patrick H
authors.initials: PH
authors.lastname: Brown
authors.forename: Marc S
authors.initials: MS
authors.lastname: Lewis
authors.forename: Chad A
authors.initials: CA
authors.lastname: Brautigam
authors.forename: Peter
authors.initials: P
authors.lastname: Schuck
authors.forename: Huaying
authors.initials: H
authors.lastname: Zhao
mesh_terms: D019540:Area Under Curve; D002138:Calibration / Q000592:standards*; D015203:Reproducibility of Results; D012710:Serum Albumin, Bovine / Q000032:analysis*; D013696:Temperature; D013995:Time; D014461:Ultracentrifugation / Q000295:instrumentation / Q000379:methods* / Q000592:standards
chemical_list: D012710:Serum Albumin, Bovine
keywords: Hydrodynamic modeling; Sedimentation equilibrium; Sedimentation velocity