We have recently expanded our light scattering service by implementing DYNAMIC LIGHT SCATTERING instrumentation. Although this approach does not provide the precision possible with static light scattering detection, the measurement requires small amounts of protein (minimal amount for a protein of MW ~40 kDa is 25 microliters of a sample with ~0.5 mg/ml). The measurement is faster than during static light scattering measurement and, most importantly, sample does not experience any dilution during measurement.
The DLS experiment measures time fluctuations of light intensity caused by motions of macromolecules in solution. How rapidly the intensity fluctuates over time is represented by an autocorrelation function. Analysis of the correlation function decay as a function of short time intervals can be used to evaluate the translational diffusion coefficient, D. The hydrodynamic radius, Rh, can be then calculated from D using the Stokes-Einstein equation. Additionally, the heterogeneity of the sample can be assessed by mathematical manipulations of the autocorrelation function. A simple way of representing heterogeneity is the polydispersity factor, which should be small (less than ~ 10%) for homogeneous sample. If the sample is heterogeneous, it is possible (at least in principle) to obtain a distribution of Rh through mathematical manipulation of autocorrelation function.
Molecular weight, MW, is calculated from the diffusion coefficient using a calibration plot of logD versus logM. This calibration curve is provided by the manufacturer of the instrument based on the Rh measurement for well characterized proteins of known D and MW.
Although molecular weights can be determined also via mass spectrometry and analytical centrifugation, only light scattering and analytical centrifugation monitor the properties of macromolecules in solution and provide information about the oligomeric state of the protein. The comparatively short time of an DLS measurement greatly facilitates carrying out the multiple studies that may be needed to determine the impact of protein concentration, ligands, pH.
The Biophysical Resource at Keck Facility offers the Dynamic Light Scattering Detector (DynaPro, Wyatt Technology - formerly Protein Solutions) as an "open access" instrument that can be used by trained users. To schedule a training session please contact Ewa Folta-Stogniew
For DLS schedule go to Scheduler
Short "start up protocol"
First time users of the resource follow the booking and charging protocol; please fill out the Biophysics Resource Usage Form from the box on the left**, and email the completely filled usage form, with email Subject: "Biophysics Resource Usage," to Ewa Folta-Stogniew.
**Attention All Mac users using "Preview" instead of "Acrobat", please save PDFs as follows: 1. Under the "File" pulldown menu, Select "Print…" 2. Click "PDF" button in lower left hand corner of window. 3. In the pulldown menu Select "Save As PDF." 4. Save PDF.**