Sedimentation Velocity

Sedimentation velocity (SV-AUC) is an analytical ultracentrifugation method that measures the rate at which molecules move in response to centrifugal force generated in a centrifuge. This sedimentation rate provides information about both the molecular mass and the shape of molecules. In some cases this technique can also measure diffusion coefficients and molecular mass.


In the biotechnology industry sedimentation velocity is used much more frequently than sedimentation equilibrium  and thus when biotech scientists say "AUC" or "analytical ultracentrifugation" they typically really mean "sedimentation velocity".

Sedimentation velocity is particularly valuable for:


  • verifying whether a sample is entirely homogeneous in mass and conformation

  • detecting aggregates in protein samples and quantifying the amount of aggregate

  • comparing the conformations for samples from different lots, manufacturing processes, or expression systems (comparability studies), or comparing different engineered variants of the same protein/peptide [see presentations in Resources]

  • establishing whether the native state of a protein or peptide is a monomer, dimer, trimer, etc.

  • determining the overall shape of non-glycosylated protein and peptide molecules in solution (are they approximately spherical or highly extended and rod-like?)

  • measuring the distribution of sizes in samples which contain a very broad range of sizes

  • detecting changes in protein conformation, for example partial unfolding or transitions to "molten globule" states

  • studying the formation and stoichiometry of tight complexes between proteins (for example receptor-ligand or antigen-antibody complexes)


In the sedimentation velocity method a sample is spun at very high speed (usually 40-60 K rpm) in an analytical ultracentrifuge. The high centrifugal force rapidly depletes all the protein from the region nearest the center of the rotor (the meniscus region at the air/solution interface), forming a boundary which moves toward the outside of the rotor with time (see example below), until finally all the protein forms a pellet at the outside of the cell. The concentration distribution across the cell at various times during the experiment is measured while the sample is spinning, using either absorbance or refractive index detection in our Beckman ProteomeLab XL-I  .


A major advantage of this method over sedimentation equilibrium  is that experiments usually require only 4-6 hours, as opposed to the several days typical of sedimentation equilibrium. Thus sedimentation velocity can be used with samples that are too labile for sedimentation equilibrium. The major drawback relative to sedimentation equilibrium applies to interacting systems (proteins that reversibly self-associate or protein-protein complexes), where the non-equilibrium nature of the measurement can lead to significant changes in species distributions over the course of an experiment. Further, for interacting systems it is generally more difficult and less accurate to derive binding constants (Kd's) from sedimentation velocity data.


An important strength of sedimentation velocity is its ability to study samples over a fairly wide range of pH and ionic strength conditions (and often directly in formulation buffers), and at temperatures from 4 to 40 °C. The amount of protein required depends on the application, but each sample is usually ~0.45 mL at typical protein concentrations of 0.1-1 mg/mL (45-450 micrograms total). Protein concentration can range as low as ~10 micrograms/mL or as high as ~40 mg/mL in some cases (but generally the concentration should be 2 mg/mL or below). Up to 3 samples can be run at one time.


A sedimentation velocity case study: a monoclonal antibody

To better illustrate this technique and some of its uses, some data for a monoclonal antibody will be presented here and the interpretation of that data briefly discussed. Even more example applications for antibodies can be found among the published articles, talks, and posters marked with the symbol on our Resources page.


The graph below shows scans across the centrifuge cell, recording the absorbance at 280 nm versus position within the cell. These scans were taken starting at 13 minutes after initiating a run at 45,000 rpm (the black data set in the graph), and then every ~12 minutes thereafter (blue, green, cyan, etc.). The sharp vertical spike at 6.02 cm indicates the position of the air-solution meniscus. In the first data set the sedimentation of the antibody has already depleted its concentration in the region near the meniscus and formed a sedimentation boundary. At later times in the run the depleted region expands and the boundary moves away from the center of the rotor, until by the time of the last data set the concentration of antibody has dropped to essentially zero throughout the upper half of the cell.

The rate at which the sedimentation boundary moves is a measure of the sedimentation coefficient of the protein. The sedimentation coefficient depends on the molecular weight (larger proteins sediment faster) and also on molecular shape. Unfolded proteins or one with highly elongated shapes will experience more hydrodynamic friction, and thus will have smaller sedimentation coefficients than a folded, globular protein of the same molecular weight.


The minimum width of the sedimentation boundary is related to the diffusion coefficient of the molecule; the presence of multiple species with similar sedimentation coefficients will cause the boundary to be broader than expected on the basis of diffusion alone. In this case the majority of the boundary is reasonably narrow, but the slow rise of the data on the right side of the boundary suggests the presence of some faster moving species.


Today the raw SV-AUC data are often processed to yield a sedimentation coefficient distribution using the "c(s) method", as shown in the figure below. This distribution resembles a chromatogram, and in many ways is similar to a size-exclusion chromatogram, except the peaks come in the opposite order. Like a chromatogram, the area under each peak gives the total amount of that species.


In this figure the full distribution is shown in the main graph, while the inset magnifies the vertical scale by 10X in order to better show the minor components. With the enhanced resolution we now see a fully baseline-resolved dimer peak at ~9.4 S (4.7% of the total protein), and small peaks at ~13.7 S and 17.5 S (1.8% and 1.1%, probably trimer and tetramer). In addition there is 0.7% of a low mass contaminant at 2 S (possibly a Fab-like fragment or free light chain). The fitting procedure also tells us that the molar mass of the main peak is about 150 kDa, consistent with assigning that peak as the antibody monomer.


Thus from this one velocity experiment, we have been able to quantify the amount and mass of the main component, the content and sedimentation coefficients of 3 aggregates and a low mass contaminant, and to obtain information about the conformation of the main component.


To further illustrate the excellent resolution and sensitivity that can be obtained, the next figure shows results for a highly stressed sample of another monoclonal antibody. We see well-resolved peaks for dimer, trimer, ... out to heptamer, as well as 2 peaks for fragments. (The hexamer and heptamer peaks are too small to see on this scale.) This resolution is much better than can be achieved by size-exclusion chromatography, and a much broader range of sizes can be covered in one experiment via sedimentation velocity (this analysis could easily have been extended out to masses ~10-fold higher).

Other applications

The figure below shows a sedimentation coefficient distribution for a sample of adeno-associated virus (AAV), used to deliver vectors for gene therapy. This illustrates the broad range of sizes and molecule types that can be studied using this technique. The inset shows a 400-fold expanded scale to allow the many minor aggregate peaks to be seen. The main peak at 106 S represents the fully-assembled virus monomer (the desired product, which is only about 80% of the total in this case). The first peak at 66 S represents empty capsids (containing no packaged DNA vector), and the second peak at 88 S represents half-full capsids (containing only 1 copy of the genome rather than 2).

Note that the assignments of the first few aggregate peaks as dimer, trimer, and tetramer of the fully-assembled virus are uncertain, but the sedimentation coefficients of those minor peaks fall within the range expected for those oligomers.

We have also used SV-AUC to characterize several other viruses, and many other types of molecules including nucleic acids, carbohydrates, carbon nanotubes, lipid-based adjuvants for vaccines, and more.