A simple cell-alignment protocol for sedimentation velocity analytical ultracentrifugation to complement mechanical and optical alignment procedures

Abstract

In establishing the sources of data variability within sedimentation velocity analysis in the analytical ultracentrifuge and their relative importance, recent studies have demonstrated that alignment of the sample cells to the centre of rotation is the most significant contributing factor to overall variability, particularly for the characterisation of low levels of protein aggregation. Accurate mechanical and optical alignment tools have been recently designed. In this study, we (1) confirm the effect of misalignment observed by others on the estimated amounts of bovine serum albumin (BSA) monomer and dimer, and the sedimentation coefficient value for the BSA dimer; and (2) demonstrate the high performance of a mechanical alignment tool and the usefulness of a simple and complementary enhanced manual alignment protocol which should be useful for situations where these tools are not available.

Keywords: Dimerisation; Improving measurement precision; Protein aggregation.

Fig. 1

Nanolytics cell alignment tool. Courtesy of Dr. K. Schilling

Fig. 2

Cell misalignment tool. a In position at the base of the rotor, b cells after misalignment. 1,2 alignment lines, 3 locating groove

Fig. 3

Mechanical tool aligned 0° data. Sedimentation coefficient distribution plot for 0.7 mg/mL bovine serum albumin in phosphate–chloride buffer (pH 6.8, I = 0.1). Alignment with Nanolytics mechanical tool set for 0°. I: 1st run. II: 2nd run after roller re-dispersal. III. 3rd run at 0° after completion of all the misalignment and re-dispersal experiments. IV: Enhanced manual alignment, 0°

Fig. 4

Effect of misalignment. a Sedimentation coefficient distribution plot for 0.7 mg/mL bovine serum albumin in phosphate–chloride buffer (pH 6.8, I = 0.1) for a range of misalignment angles. b Monomer peak height as a function of misalignment angle. The average value and standard error for 0° is shown and is assumed by % the same for the misaligned angles

Fig. 5

Relative percentages (%) of estimated BSA monomer and dimer as a function of misalignment angle. The average value and standard error for 0° is shown and is assumed by % the same for the misaligned angles