Optical alignment procedure for the analytical ultracentrifuge

Gropper, L.

doi:10.1016/0003-2697(64)90152-6

Cited by 58 publications

(7 citation statements)

References 6 publications

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“…As pointed out previously [20], the graphical determination traditionally required for pre-computer analysis methods introduces errors for several reasons [20,59], including the fact that the width of the optical artifact is relatively broad when compared to the precision of the computational determination of this parameter [26]. Although none of the data sets in this study showed any evidence for convection, subtle influences of this factor can be strongly diminished by adding the degree of freedom for the meniscus position to the analysis.…”

Section: Resultsmentioning

confidence: 99%

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

Ghirlando

Balbo

Piszczek

et al. 2013

Analytical Biochemistry

106

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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.

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Section: Resultsmentioning

confidence: 99%

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

Ghirlando

Balbo

Piszczek

et al. 2013

Analytical Biochemistry

106

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show abstract

“…as was studied in detail previously by several investigators (Trautman 1958 ; Erlander and Babcock 1961 ; Gropper 1963 ). Gropper has pointed out that with the focus of the optics being at the 2/3 plane of the solution column, the meniscus position will not appear at the correct image location (Gropper 1964 ). As emphasized by Philo ( 1997 ), “it is not entirely clear how to determine the true correct meniscus position from the experimental data.”…”

Section: Discussionmentioning

confidence: 99%

“…Finally, we addressed what currently seem to be two major experimental limitations, one being the possible effect of low level convection on the sedimentation process, and one arising from the inability to determine experimentally the precise meniscus position. The difficulty of determining the meniscus position is due to both the presence of significant local optical artifacts at the liquid/air interface and the limited radial resolution of the experimental scans (Gropper 1964 ; Philo 1997 ; Pekar and Sukumar 2007 ). As has been emphasized by Philo ( 1997 ), it is not clear how to determine the true meniscus parameter, yet it seems an essential model parameter for predicting the boundary position.…”

Section: Introductionmentioning

confidence: 99%

On the analysis of sedimentation velocity in the study of protein complexes

2009

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Sedimentation velocity analytical ultracentrifugation has experienced a significant transformation, precipitated by the possibility of efficiently fitting Lamm equation solutions to the experimental data. The precision of this approach depends on the ability to account for the imperfections of the experiment, both regarding the sample and the instrument. In the present work, we explore in more detail the relationship between the sedimentation process, its detection, and the model used in the mathematical data analysis. We focus on configurations that produce steep and fast-moving sedimentation boundaries, such as frequently encountered when studying large multi-protein complexes. First, as a computational tool facilitating the analysis of heterogeneous samples, we introduce the strategy of partial boundary modeling. It can simplify the modeling by restricting the direct boundary analysis to species with sedimentation coefficients in a predefined range. Next, we examine factors related to the experimental detection, including the magnitude of optical aberrations generated by out-of-focus solution columns at high protein concentrations, the relationship between the experimentally recorded signature of the meniscus and the meniscus parameter in the data analysis, and the consequences of the limited radial and temporal resolution of the absorbance optical scanning system. Surprisingly, we find that large errors can be caused by the finite scanning speed of the commercial absorbance optics, exceeding the statistical errors in the measured sedimentation coefficients by more than an order of magnitude. We describe how these effects can be computationally accounted for in SEDFIT and SEDPHAT.

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“…These solutions were examined in a Beckman Spinco model E ultracentrifuge equipped with both schlieren and Rayleigh-interference optical systems. The optical systems had been accurately aligned by methods essentially the same as those described by Gropper (1964). Sedimentation-velocity studies were done at 20°C and at 59 780rev./min by using schlieren optics.…”

Section: Methodsmentioning

confidence: 99%

Sedimentation-equilibrium studies on the heterogeneity of two β-lactamases

Lloyd

Peacocke

1970

Biochemical Journal

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Solutions of crystalline beta-lactamase I and beta-lactamase II, prepared by Kuwabara (1970), were examined in the ultracentrifuge and their sedimentation coefficients, diffusion coefficients, molecular weights and heterogeneity determined. Each sample was shown to consist of a major component comprising at least 97% of the material and a minor component of much higher molecular weight. The molecular weights of the major components were 27800 for beta-lactamase I and 35600 for beta-lactamase II. Emphasis is placed on a straightforward practical way of analysing the sedimentation-equilibrium results on mixtures of two macromolecular components rather than on a strict theoretical solution. Appendices describe the theory of systems at both chemical and sedimentation equilibrium and the procedure for calculating the combined distribution of two components.

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Optical alignment procedure for the analytical ultracentrifuge

Cited by 58 publications

References 6 publications

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

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

On the analysis of sedimentation velocity in the study of protein complexes

Sedimentation-equilibrium studies on the heterogeneity of two β-lactamases

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