Investigation of protein three dimensional structures based on recent methods, such as nuclear magnetic resonance spectrometry, (NMR), small-angle X-ray scattering (SAXS), cryo-electron microscopy (cryo-EM) and X-ray crystallography, relies on the absence of protein aggregates. Therefore sample preparation plays a crucial role for the successful application of all these methods. Since each step of sample preparation implies the risk to create aggregates, verification of the aggregation state after each step enables fast identification of weak points and contributes significantly to optimize sample preparation protocols. Such an analysis tool is dynamic light scattering (DLS) which has already many applications (Berne & Pecora, 1976). Key features of DLS analysis tools is a minimal sample volume, ease of use and usefulness of output. DLS applied in situ fulfills these requirements.
In this lecture in situ DLS measurements on sample aliquots of ~1 µl in multiwell plates (hanging drop, sitting drop or under oil) will be demonstrated, using a fully automated instrument. In order to demonstrate the capabilities of this device, model protein radius distributions will be measured. Measurements will be applied on aliquots of protein solutions as examples of intermediates from the daily lab work taken from purification steps e.g. concentration increasing. Another application is the analysis of crystallizing samples, before and after precipitant addition. Signal interpretation allows determination of aggregation or nucleation indicating the position in the phase diagram (Vekilov, 2010). Determination of micelle sizes is also easily feasible. This may be used to identify protein detergent complexes after addition of membrane proteins. In addition, a built-in camera allows to observe all wells, this means the instrument can be used as an imaging system as well (Meyer 2014). In combination with a UV light source it is even possible to distinguish protein from salt crystals by using the intrinsic fluorescence of proteins containing tryptophan.