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Sample Preparation Guide

In order to perform a successful analysis, it is important that the sample shipped to our facility is in optimal condition. The following issues should be considered when preparing samples for AUC analysis:

• Buffer Absorbance:

  When preparing samples for analysis using UV or visible absorption measurement, it is important that the buffer does not absorb at the wavelength where the sample is measured. To make sure that the buffer does not absorb, measure your buffer's absorbance against water at the wavelength of measurement. When in doubt, use a non-absorbing buffer like sodium or potassium phosphate. TRIS is OK at 280 nm.

• Salt:

  Salt often helps to make a sample behave more ideal in solution. We suggest a minimum of 100-200 mM NaCl. It often prevents aggregation for charged molecules or helps to prevent other nonideality effects.

• 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.

• Fluorescent Experiments:

  Fluorescence experiments measure the intensity of fluorescently tagged molecules. Excitation is at 488 nm so that tags such as Alexa488, SybrGreen, fluorescein, and other fluorescein based tags can be used. Samples must be degassed prior to analysis and the use of a non-tagged carrier protein such as ovalbumin should be considered especially for proteins with very low concentrations.

• Rayleigh Interference Experiments:

  Interference experiments measure the refractive index gradient between a sample and a reference. Since all buffer components contribute to the refractive index of the sample, it is important that the concentration of all buffer components is exactly equal on both sample and reference sector. The only way to assure that the contribution of salts and other buffercomponents is identical on both sides is through either extensive dialysis or by using the column eluate from the protein purification as reference buffer.

• 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:

  1. insufficient salt for charged proteins
  2. hydrophobic regions in the protein exposed to aqueous buffers
  3. incorrect pH
  4. incorrect salts in buffer
  5. bi-polar proteins
  6. 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.