Bitan:Dynamic Light Scattering
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<o:DocumentProperties> <o:Author>Bernhard Monien User</o:Author> <o:Template>Normal</o:Template> <o:LastAuthor>HL</o:LastAuthor> <o:Revision>2</o:Revision> <o:TotalTime>0</o:TotalTime> <o:Created>2009-11-26T07:31:00Z</o:Created> <o:LastSaved>2009-11-26T07:32:00Z</o:LastSaved> <o:Pages>1</o:Pages> <o:Words>578</o:Words> <o:Characters>3300</o:Characters> <o:Lines>27</o:Lines> <o:Paragraphs>6</o:Paragraphs> <o:CharactersWithSpaces>4052</o:CharactersWithSpaces> <o:Version>11.1282</o:Version> </o:DocumentProperties> <o:OfficeDocumentSettings> <o:AllowPNG/> </o:OfficeDocumentSettings>
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#E6E6E6;border-collapse:collapse;mso-padding-alt:0in 5.4pt 0in 5.4pt'> <tr> <td width=868 valign=top style='width:868.0pt;padding:0in 5.4pt 0in 5.4pt'> <p class=MsoNormal align=center style='text-align:center'><span style='font-size:16.0pt;color:#993300'><b>Dynamic light scattering <o:p></o:p></b></span></p> <p class=MsoNormal align=center style='text-align:center'><span style='color:black'><b><![if !supportEmptyParas]> <![endif]><o:p></o:p></b></span></p> <p class=MsoNormal><span style='color:black'>Precision Detectors PD2000DLS <o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>Quasielastic light scattering (QLS) spectroscopy is an optical method for the determination of diffusion coefficients of particles in solution.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'><b>Instrumental procedure:<o:p></o:p></b></span></p> <p class=MsoNormal><span style='color:black'>1. Switch on computer.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>2. Switch on the laser beam PDDLS/Batch<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>3. Switch on detector PD2000DLS.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>4. Open PrecisionDeconvolve program on the desktop of computer. <o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>5. Parameter setting: <o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>5.1 Sample time– maintain at least 1 photo count per correlation time.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>5.2 Run time–<span style="mso-spacerun: yes"> </span>intervals between detector measurements. Recommend 5-20s.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>5.3 Accumulation time–<span style="mso-spacerun: yes"> </span>the amount of time over which the individual run times are averaged to produce a window with your distribution of species. Recommend 4-10.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>5.4</span><span style='color:black;mso-fareast-language:ZH-CN'> Repeat # – the number of accumulated run times you to repeated. <o:p></o:p></span></p> <p class=MsoNormal><span style='color:black;mso-fareast-language:ZH-CN'>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.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black;mso-fareast-language:ZH-CN'>6. Data is saved under measurement setup/sample record/path. <o:p></o:p></span></p> <p class=MsoNormal><span style='color:black;mso-fareast-language:ZH-CN'>7. Click GO will start measurements.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black;mso-fareast-language:ZH-CN'>8. After the measurements, the data information could be viewed at setup/ data record view.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black;mso-fareast-language:ZH-CN'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'><b>Sample preparation:<o:p></o:p></b></span></p> <p class=MsoNormal><span style='color:black'>1. </span>Dissolve compound in required buffer solution.</p> <p class=MsoNormal>2. Centrifuge at 5000 g for 30 minutes to remove <span style='color:black'>the big undissolved materials or dust.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>3. Transfer the CLEAN (dust free) solution to Kimble disposable culture glass tubes (6×50 mm), which is clean and fit to sample holder. <o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>4. Test DLS to see if the concentration is enough for recording.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'><b>Notes for sample preparation:<o:p></o:p></b></span></p> <p class=MsoNormal><span style='color:black'>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.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>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.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>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.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>4. Centrifugation method is less effective in removing large flaky particles or linear aggregates.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>5. Keep the sample covered at all times. This will eliminate the possibility of dust particles from the room from entering the sample.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>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.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>7. After the sample filled in the tube and sealed, clean the outside of the tube with lens paper and methanol.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>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.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>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).<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>(The method is modified from Dr. Aleksey Lomakin, Dr. Bernhard Monien, and Dr. Ghiam Yamin’s protocols)<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'><![if !supportEmptyParas]> <![endif]><o:p></o:p></span></p> <span style='font-size:12.0pt;font-family:"Times New Roman";color:black; mso-ansi-language:EN-US;mso-fareast-language:EN-US'><br clear=ALL style='page-break-before:always'> </span> <p class=MsoNormal><span style='color:black'><b>Recommended reading:<o:p></o:p></b></span></p> <p class=MsoNormal><span style='color:black'>Aleksey Lomakin, David B. Teplow, and George B. Benedek. Quasielastic Light Scattering for Protein Assembly Studies. <i>Methods in Molecular Biology, vol. 299: Amyloid Proteins: Methods and Protocols. </i></span><span style='color:black'>Edited by: E. M. Sigurdsson © Humana Press Inc., Totowa, NJ.<o:p></o:p></span></p> <p class=MsoNormal><span style='color:black'>Aleksey Lomakin and David B. Teplow. Quasielastic Light Scattering Study of Amyloid b-Protein Fibril Formation. <i>Protein & Peptide letters, </i></span><span style='color:black'>2006, 13, 247–254.<o:p></o:p></span></p> <p class=MsoNormal align=center style='text-align:center'><![if !supportEmptyParas]> <![endif]><span style='color:black'><b><o:p></o:p></b></span></p> </td> </tr>
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