Ensuring Optimal Sample Condition

It is important that any samples set to a facility is sent in optimal condition. The following issues must be considered.

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.

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.

Microfuge Test

The Microfuge Test is a simple test that can be performed to make sure that your sample is not aggregating. This test should be performed on all samples prior to shipping. The following steps are involved:

  1. Prepare 1 ml of a high concentration (4-5 mg/ml) sample of your protein.

  2. Measure the absorbance at 280 nm (or 230 nm, if there is no absorbance at 280). Dilute as necessary. Never trust a measurement above 0.9 OD (nonlinearity of optics!).

  3. Spin sample for 20 minutes in microfuge.

  4. Carefully aspirate a small amount of the supernatant, without disturbing the solution.

  5. Measure the OD. If the OD is reduced, the sample is aggregating.

  6. If the sample is OK, repeat the microfuge test after storing it for 2-3 days at the same temperature at which the AUC experiment will be performed.

If the OD of the supernatant does not change over time, the sample is suitable for AUC analysis.