Measurements

General considerations for sample prep:

  • As a rule of thumb, the concentration of the samples should be:

  • We have 2 cuvettes available: 1 mm path length (350 μL) and 0.1 mm path length (27 μL).

  • Sample purity should be ≥ 95% (can be determined by HPLC, gel electrophoresis or mass spec).

  • An accurate measurement of the protein concentration is very important with CD. It is recommended to check the concentration right before conducting the experiment to account for changes in concentration due to precipitation etc. A reliable and quick way to do so is by measuring the absorbance at 280 nm. Other methods are described in the Greenfield review.

  • Sample absorbance should stay below 2.0 AU, with signal-to-noise ratios obtained when total sample absorption is between 0.6 and 1.2 AU (the most optimal absorbance value is ~ 0.8 AU according to the manual). If the absorbance is above 2.0 AU, the sample must be diluted.

  • The HV value also should not drop below 200 V when using a bandwidth of 1 nm. If the HV drops below this threshold then the bandwidth needs to be reduced.

Measurements:

Once the lamp has warmed up, you can start collecting data.

  • Secondary structure determination wavelength range (far UV): 170 - 240 nm

  • Tertiary structure environment (near UV): 260 - 300 nm

The suggested parameters are:

General sequence:

  1. Configure the acquisition settings as required—some preliminary work may be required to establish the optimal conditions.

  2. Acquire a background. The background enables you to measure the absorbance spectrum of your buffer and sample.

      1. Mount the cuvette holder in the Single Cell Peltier Holder, but do not insert a cuvette.

      2. Click Background on the Background panel .

  3. Acquire a sample CD spectrum .

      1. Load the cuvette containing sample into the Single Cell Peltier Holder.

      2. Set the file name for the sample.

      3. Click Acquire on the Sequencer panel.

  4. Acquire a buffer CD spectrum .

      1. Remove and clean the cuvette and fill with the buffer, reload the cuvette into the Single Cell Peltier Holder.

      2. Set the file name for the buffer.

      3. Click Acquire on the Sequencer panel.

Rinsing the cuvette in between runs:

Using 3 x detergent, then 3 x H2O, then 95% EtOH. Dry using the N2 cylinder (refer to the image).