How do you choose the best buffer for sedimentation velocity experiments?

1 Buffer composition

The buffer composition affects the viscosity, density, refractive index, and pH of the solution, which in turn affect the sedimentation coefficient and the optical detection of the sample. You want to choose a buffer that minimizes these effects and maintains the stability and solubility of your sample. A common choice is phosphate-buffered saline (PBS), which has a moderate ionic strength, a neutral pH, and a low viscosity. However, you may need to adjust the buffer composition depending on your sample properties and your analytical goals. For example, you may want to add salt, detergent, glycerol, or other additives to modulate the interactions, aggregation, or hydrodynamic properties of your sample.

2 Buffer concentration

The buffer concentration also influences the sedimentation behavior of your sample. A higher buffer concentration increases the viscosity and density of the solution, which reduces the sedimentation rate and the resolution of the experiment. A lower buffer concentration decreases the viscosity and density of the solution, which increases the sedimentation rate and the resolution of the experiment. However, a too low buffer concentration may compromise the stability and solubility of your sample, or cause unwanted changes in the pH or the ionic strength of the solution. Therefore, you want to choose a buffer concentration that balances these factors and provides optimal conditions for your sample and your analytical method. A typical range is 10-50 mM for the buffer concentration.

3 Buffer volume

The buffer volume affects the loading and the performance of the sedimentation velocity experiment. You want to choose a buffer volume that fills the cell adequately and prevents air bubbles from forming. Air bubbles can interfere with the optical detection and the sedimentation equilibrium of the sample. You also want to choose a buffer volume that minimizes the dilution and the loss of your sample during the loading and the centrifugation. A too large buffer volume may reduce the concentration and the signal of your sample, while a too small buffer volume may cause spillage or leakage of your sample. Therefore, you want to choose a buffer volume that matches the capacity and the design of your cell and your rotor. A typical range is 400-450 μL for the buffer volume.

4 Buffer quality

The buffer quality affects the purity and the consistency of your sedimentation velocity experiment. You want to choose a buffer that is free of contaminants, such as dust, microbes, or impurities, that can alter the properties or the behavior of your sample. You also want to choose a buffer that is freshly prepared or properly stored, to avoid degradation or changes in the composition or the pH of the solution. Therefore, you want to choose a buffer that is filtered, sterilized, and checked for its quality before use. You may also want to degas your buffer to remove dissolved air or gases that can affect the viscosity or the density of the solution.

5 Buffer validation

The buffer validation is the final step to ensure that you have chosen the best buffer for your sedimentation velocity experiment. You want to validate your buffer by testing its suitability and compatibility with your sample and your analytical method. You can do this by performing some preliminary experiments, such as measuring the viscosity, density, refractive index, and pH of your buffer, comparing the sedimentation profiles of your sample in different buffers, or checking the stability and solubility of your sample in your buffer. You can also use some software tools, such as SEDNTERP or SEDFIT, to calculate or simulate the effects of your buffer on your sedimentation coefficient and molecular weight. By validating your buffer, you can optimize your experimental conditions and improve your data quality and interpretation.

6 Here’s what else to consider

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