Improving the thermal, radial, and temporal accuracy of the analytical ultracentrifuge through external references

Rodolfo Ghirlando, Andrea Balbo, Grzegorz Piszczek, Patrick H. Brown, Marc S. Lewis, Chad A Brautigam, Peter Schuck, Huaying Zhao

Research output: Contribution to journalArticle

49 Scopus citations

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.

Original languageEnglish (US)
Pages (from-to)81-95
Number of pages15
JournalAnalytical biochemistry
Volume440
Issue number1
DOIs
StatePublished - Jan 1 2013

Keywords

  • Hydrodynamic modeling
  • Sedimentation equilibrium
  • Sedimentation velocity

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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