Ensuring Optimal Sample Condition

Buffer Absorbance

If you are preparing your samples for analysis using UV/vis absorption measurements, it is important that your buffer does not absorb at your chosen wavelength.

Measure your buffer's absorbance against water at the wavlength of measurement. Otherwise, use a non-absorbing buffer such as sodium or potassium phosphate. At 280 nm, it is okay to use TRIS.

Salt

Salt can make a sample behave more ideally in solution. A minimum of 100-200 mM NaCl can prevent aggregation for charged molecules and/or help prevent other non-ideality effects.

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Glycerol

Glycerol tends to redistribute in the ultracentrifugation cell to build up a gradient of its own, affecting the density and viscosity of the buffer throughout the cell. This is very difficult to model correctly, because it is so difficult or impossible to measure. In velocity experiments the contribution of glycerol is often quite noticeable, and should be avoided whenever possible. Low amounts of a few percent do not seem to cause any significant problems for equilibrium experiments.

Detergents

Detergents often absorb at lower wavelengths and therefore can be unsuitable for absorbance measurements at lower wavelengths. They also contribute to the partial specific volume of the observed particle and it is difficult to estimate the correct amount of bound detergent.

Aggregation

Clearly, aggregation is counterproductive to any analytical ultracentrifugation experiment and should be avoided at all cost. The approach to prevent aggregation differs from protein to protein, and the best method should be determined by the protein chemist. The following factors are known to contribute to aggregation:

  • insufficient salt for charged proteins

  • hydrophobic regions in the protein exposed to aqueous buffers

  • incorrect pH

  • incorrect salts in buffer

  • bi-polar proteins

  • presence of incorrectly formed disulfide bonds

Sometimes addition of salt, detergent, reductants or other chemicals can alleviate these problems. All samples submitted for AUC analysis should be checked for aggregation by the microfuge test prior to shipping.