Concentration Measurements
The importance of measuring accurate concentrations for velocity experiments:
When designing AUC experiments for UV optics it is highly recommended to measure in intensity rather than absorbance mode (further explanations can be found here). 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. If you are running an experiment in our facility, we will need to charge you extra time for repeating the run, reloading the samples and the time involved in determining the proper dilution for your protein.
In our experience any concentration validation measurements for sample loading performend with a Nanodrop system is prone to significant error. Therefore, all users of our facility are required to use a standard 1.0 cm cuvette on a well-calibrated UV/vis spectrophotometer. All measurements should be validated to be linear by also measuring a 1:1 dilution. Accurate OD measurements are essential! Incorrect concentrations will lead to significant additional work for us and cost to you.