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Tips for successful Absorbance/Intensity Experiments:

• A proper experimental design requires that the absorbance profile of a sample is measured before the experiment on a spectrophotometer with a 1 cm pathlength cuvette to validate the concentration of the sample (Nanodrop devices are typically not precise enough for this task). A review of the absorbance trace will reveal one or more optimal wavelength where the experiment can be measured. In the older Proteomelab XLA/XLI instruments, the monochromator is not precise enough to reset the wavelength from scan to scan to the same wavelength, and unless absorbance is constant over the measured wavelength (highly unlikely), it should not be changed mid-run. This restriction is not necessary on the newer Optima AUC models.
• Based on the pattern in the absorbance trance, it may be necessary to adjust the concentration or the wavelength until a place is found where the sample absorbs optimally (0.3-0.9 OD, with 0.6 OD typically providing the best signal-to-noise ratio and the highest linearity range). It is also important that the dynamic range of the absorbance detector is not "wasted" on background absorbance from the buffer. Therefore, always make sure the buffer (as measured against ddH2O) does not contribute to the absorbance at the wavelength selected for measurement. Because of the imprecision of the monochromator, the wavelength cannot be changed during the experiment in the XLA/XLI instruments and if several samples are to be measured at different concentrations, they still must be measured at the same wavelength. This may require that samples are sorted by absorbance and are run separately, each set at a different wavelength.
• Running a wavelength absorbance scan on the sample and buffer from 220-300 nm (for most proteins without a visible chromophore, otherwise a larger range) will provide important information for the experimental design and is highly worthwhile to assure optimal data collection.
• Both buffer and sample should always be measured against ddH2O to reveal background absorbance in the buffer. Unless the sample is extremely dilute or in an absorbing buffer, one will always be able to find a suitable wavelength for the experiment in this range. If multiple wavelengths are suitable for a sample, a wavelength should be selected that has a large emission intensity (review the the wavelength emission profile of the Xenon flash lamp), because this will provide more light to the photomultiplier tube and improve data quality and extend dynamic range.

Summary:

• Never change the wavelength midrun in the Proteomelab XLA/XLI instruments when measuring velocity experiments
• Always measure an extinction spectrum of your sample in spectrophotometer fitted with a 1 cm pathlength cuvette (Nanodrop is too unreliable/imprecise)
• Select a wavelength between 0.3-0.9 OD, with 0.6-0.7 OD typically providing the best signal-to-noise ratio.