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

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• 作者：Rodolfo Ghirl ; o ; Andrea Balbo ; Grzegorz Piszczek ; Patrick H. Brown ; Marc S. Lewis ; Chad A. Brautigam ; Peter Schuck ; Huaying Zhao
• 关键词：Sedimentation velocity ; Sedimentation equilibrium ; Hydrodynamic modeling
• 刊名：Analytical Biochemistry
• 出版年：2013
• 出版时间：1 September, 2013
• 年：2013
• 卷：440
• 期：1
• 页码：81-95
• 全文大小：2116 K

文摘

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.