If the sample cannot be concentrated and the signal is not sufficient for a reliable measurement, the user should adopt a setup equipped with a more powerful laser. This will yield the best signal at a given sample concentration. This means that the difference between refractive indices should be the largest. If multiple solvents meet the criteria above, the solvent that yields the largest contrast between sample and solvent should be chosen. the solvent should not dissolve the particles. Secondly, the solvent should not alter the properties of the samples, e.g. Ensure that the chosen solvent, along with the dispersing agent, yields sufficient colloidal stability over a time span that allows for DLS measurements. In some cases, it might be necessary to use dispersing agents such as steric or ionic surfactants. Firstly, the solvent should disperse the particles well. In order to choose a solvent, two main requirements should be taken into account. In the case of dry samples (powders), solvent addition is necessary prior to measurement. After production or synthesis and purification if prepared in a research lab, in most cases samples should be already in dispersed form. Samples used for DLS should be, in general, dispersed in a liquid phase. Dry powders (these can be measured only after homogenous dispersion in the solvent).Solid particles, polymers, and proteins dispersed in a solvent.This restricts the range of samples that can be measured with this method.ĭLS allows measurement of colloids, such as: The measurement principle of Dynamic Light Scattering (DLS) is based on the Brownian motion of particles. Save PDF.Experimental Guidelines - DLS Sample Preparation I) Sample In the pulldown menu Select "Save As PDF." 4. Click "PDF" button in lower left hand corner of window. Under the "File" pulldown menu, Select "Print…" 2. **Attention All Mac users using "Preview" instead of "Acrobat", please save PDFs as follows: 1. To schedule a training session please contact Ewa Folta-Stogniewįirst 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. 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. 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. 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.Īlthough 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. Molecular weight, MW, is calculated from the diffusion coefficient using a calibration plot of logD versus logM. If the sample is heterogeneous, it is possible (at least in principle) to obtain a distribution of Rh through mathematical manipulation of autocorrelation function. A simple way of representing heterogeneity is the polydispersity factor, which should be small (less than ~ 10%) for homogeneous sample. Additionally, the heterogeneity of the sample can be assessed by mathematical manipulations of the autocorrelation function. The hydrodynamic radius, Rh, can be then calculated from D using the Stokes-Einstein equation. Analysis of the correlation function decay as a function of short time intervals can be used to evaluate the translational diffusion coefficient, D. How rapidly the intensity fluctuates over time is represented by an autocorrelation function. The DLS experiment measures time fluctuations of light intensity caused by motions of macromolecules in solution. The measurement is faster than during static light scattering measurement and, most importantly, sample does not experience any dilution during measurement. 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). We have recently expanded our light scattering service by implementing DYNAMIC LIGHT SCATTERING instrumentation.
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