AUC Experimental Design Checklist

  1. Ensure the instrument is in good-working condition, with all calibrations properly recorded.

  2. Be aware of buffer absorbance, concentration selection

    a. What are the analyte properties? sedimentation coefficenint, diffusion, partial specific volume.

    b. What are the buffer properties? Viscosity, density

    Together, this will provide information on rotor speed, and length of experiment.

  3. Column height, temperature considerations, speed selection, and experiment length.

Concentration Measurements

When designing AUC experiments for UV optics it is highly recommended to measure in intensity rather than absorbance mode. In intensity experiments one needs to make very accurate concentration measurements to avoid accidental gain set changes in the Beckman XL-A instruments. The reference channel should be filled with a 0.3 OD solution, and a 0.9 OD solution in the sample channel (both channels as determined in a 1.0 cm pathlength cuvette). In the XLA, typically a 1.2 cm pathlength centerpiece is used, which then increases absorbance to 0.36 and 1.08 OD respectively. Those levels are within the limits of linear detection on a well functioning XLA spectrophotometer at most wavelengths accessible with this instrument.

However, if the OD level in the reference channel is exceeded, gain set adjustments are likely to occur over the course of the experiment, leading to problematic data. This requires stopping the run, diluting the sample to an appropriate level, and restarting the run.

Designing Fluorescence Binding Titration Experiments

When you perform a binding experiment in fluorescence mode, the object is to follow the labeled component and measure the concentration of the free and complexed fluorescently labeled component. Of course, in a fluorescence experiment you cannot see the unlabeled component, and this makes interpretation a lot easier. The unlabeled component only becomes visible indirectly, when the labeled component is complexed with it. So we will design the experiment to see varying amounts of labeled free and complexed material.