Bitan:Dynamic Light Scattering
Dynamic light scattering
Precision Detectors PD2000DLS
Quasielastic light scattering (QLS) spectroscopy is an optical method for the determination of diffusion coefficients of particles in solution.
1. Switch on computer.
2. Switch on the laser beam PDDLS/Batch
3. Switch on detector PD2000DLS.
4. Open PrecisionDeconvolve program on the desktop of computer.
5. Parameter setting:
5.1 Sample time– maintain at least 1 photo count per correlation time.
5.2 Run time– intervals between detector measurements. Recommend 5-20s.
5.3 Accumulation time– the amount of time over which the individual run times are averaged to produce a window with your distribution of species. Recommend 4-10.
5.4 Repeat # – the number of accumulated run times you to repeated.
5.5 Cutoff intensities – the limit that the detector will ignore measurements that exceed a certain percentage of the average. Recommend 20% of the average. This value can be changed based on the size and relative stability of the scattering species.
6. Data is saved under measurement setup/sample record/path.
7. Click GO will start measurements.
8. After the measurements, the data information could be viewed at setup/ data record view.
1. Dissolve compound in required buffer solution.
2. Centrifuge at 5000 g for 30 minutes to remove the big undissolved materials or dust.
3. Transfer the CLEAN (dust free) solution to Kimble disposable culture glass tubes (6×50 mm), which is clean and fit to sample holder.
4. Test DLS to see if the concentration is enough for recording.
Notes for sample preparation:
1. The concentration of the sample required for successful measurement is dependent on the size of the scattering particles. Since a large particle will scatter more light than a smaller particle, a lower concentration will be required. If the concentration is too high, multiple scattering can be a problem. In addition, inter-particle interactions makes analysis of the results obtained at high concentration difficult.
2. It is critical that the sample and the cuvette are clean and there is no particulate matter in the sample. Dust, undissolved sample, and any extraneous matter will significantly affect the accuracy of the results.
3. Except for centrifugation method to remove the pre-aggregated protein or dust, filter filtration (20-nm pore-size anatop filter) or flow-through filtering (SEC) also could be used.
4. Centrifugation method is less effective in removing large flaky particles or linear aggregates.
5. Keep the sample covered at all times. This will eliminate the possibility of dust particles from the room from entering the sample.
6. Use the highest quality solvents available to wash cuvettes if needed (e.g. deionized or distilled water). Our tube is disposable tube, which is dust free. You need not wash it.
7. After the sample filled in the tube and sealed, clean the outside of the tube with lens paper and methanol.
8. Hold cuvettes only by the top of the cuvette and make sure that you have not deposited any fingerprints on the sides of the cuvette. Fingerprints will scatter laser light and create scatter signals that are not related to your sample.
9. As you fill the cuvette, take care to ensure that no air bubbles are created. Air bubbles will scatter light (and may indicate that the cuvette is not clean).
(The method is modified from Dr. Aleksey Lomakin, Dr. Bernhard Monien, and Dr. Ghiam Yamin’s protocols)
Aleksey Lomakin, David B. Teplow, and George B. Benedek. Quasielastic Light Scattering for Protein Assembly Studies. Methods in Molecular Biology, vol. 299: Amyloid Proteins: Methods and Protocols. Edited by: E. M. Sigurdsson © Humana Press Inc., Totowa, NJ.
Aleksey Lomakin and David B. Teplow. Quasielastic Light Scattering Study of Amyloid b-Protein Fibril Formation. Protein & Peptide letters, 2006, 13, 247–254.