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, which 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., doi 10.1016/j.ab.2013.02.011) 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 eleven instruments displayed a significantly reduced standard deviation of ∼ 0.7 %. This study demonstrates the need for external calibration procedures and regular control experiments with a sedimentation coefficient standard.

Improved measurement of the rotor temperature in analytical ultracentrifugation

“…Temperature measurements were carried out using Maxim Thermochron iButtons (Maxim Integrated Products, San Jose, CA, USA) as described [12]. Briefly, DS1922L iButtons with 11-bit resolution, corresponding to 0.0625 °C increments, and a range from −10 °C to +65 °C, were calibrated with a NIST-certified reference thermometer P795 (ThermoWorks, Lindon, UT, USA) accurate to ± 0.015 °C.…”

“…This contrasts to the precision of ~0.1% when data are acquired side-by-side in the same experiment and on the same instrument [12,13]. To account for this huge discrepancy, we developed methods for the external calibration of scan time, radial magnification, and temperature, and demonstrated that the combined application of corrections from these experimental dimensions leads to a precision equivalent to that observed for a single instrument.…”

“…In a recent study comparing ten different analytical ultracentrifuges, we found that the sedimentation coefficient of monomeric bovine serum albumin (BSA) differed by as much as 15%, depending on the instrument used [12]. This contrasts to the precision of ~0.1% when data are acquired side-by-side in the same experiment and on the same instrument [12,13].…”

“…We have introduced the use of a miniaturized integrated circuit with onboard memory (Maxim Thermochron iButton ™ ) that can log the rotor temperature as a function of time and found that measured temperatures for different centrifuges, at a set point of 20 °C, spanned a range of more than 4 °C, accounting for errors in excess of 10% in the hydrodynamic parameters. We have independently validated the correctness of the temperature readings through a sedimentation coefficient standard under control of other factors [12]. …”

Chemical and Biophysical Approaches for Complete Characterization of Lectin–Carbohydrate Interactions

“…Sedimentation data are first processed in REDATE 1.0.1 (Ghirlando et al, 2013; Zhao, Ghirlando, Piszczek, Curth, Brautigam et al, 2013) to correct for time-stamp errors and remove truncated scans.…”