The DynaPro™ Titan DLS instrument was bought in our department primarily for studying the multimeric status of different proteins. We primarily procured this instrument because in addition to being a very sensitive DLS instrument, it could also operate with the smallest sample volume: approximately 10 microliters. The first feature that fascinated us about this instrument was the size – it is extremely compact and occupies very little space. The instrument however requires an absolutely dust-free environment, as measurements are extremely dust-sensitive.
The instrument is provided with a 10 microliter cuvette for sample measurements. However, disposable cuvettes are also available that can be used in these experiments. The unit can operate at various temperatures ranging from –4 degrees Celsius to +60 degrees Celsius. The set temperature is achieved almost immediately after starting the instrument. The machine uses a laser with a wavelength of approximately 800 nm as the light source. There are also provisions for connecting a flow-cell to the instrument so that it can be linked up to a liquid chromatography system for online measurements. A chromatography system can be integrated with this instrument for real time measurements. We occasionally use this option to assess the stability of the multimeric forms of our proteins.
The instrument is provided with a nanofilter kit. This kit has filters and a small filtration unit for filtering protein samples. This is absolutely essential, as even small amounts of dust particles can give spurious results. Sometimes, we find that excessive filtration is required to clean the samples from both soluble and insoluble aggregates, as these particles interfere with the measurements. The samples used for the instrument should also be as dilute as possible, as concentrated proteins do not yield good results. The machine can be used to calculate the hydrodynamic radius of proteins as well as the molecular weight. We have measured molecular weights up to 180 kDa with this system.
Before the start of any experiment, a blank measurement with plain water is taken to check whether the cuvette is clean. If the reading does not read as water, then the cuvette should be extensively cleaned until the desired blank reading is obtained. This can sometimes be a tedious process, as the cuvette is very small. Beginners should practice with BSA (bovine serum albumin) until consistent data is obtained. We have observed that with practice, accuracy of the data obtained increases.
The instrument can be used to measure protein dimensions in any buffer of choice, provided the refractive index of the buffer is known beforehand. We use phosphate buffered saline for all our experiments. Glycerol is mostly avoided in the buffer system. Once standardization is achieved, the experiment is generally complete in less than one hour.
The instrument is completely computer-controlled; the software used for the complete operation is Dynamics Pro. We have also noticed that although the software provided is easy to use and the manual is quite descriptive, an initial training from an expert is very helpful.