<html xmlns:v="urn:schemas-microsoft-com:vml" xmlns:o="urn:schemas-microsoft-com:office:office" xmlns:w="urn:schemas-microsoft-com:office:word" xmlns:m="http://schemas.microsoft.com/office/2004/12/omml" xmlns="http://www.w3.org/TR/REC-html40"><head><meta http-equiv=Content-Type content="text/html; charset=us-ascii"><meta name=Generator content="Microsoft Word 14 (filtered medium)"><style><!--
/* Font Definitions */
@font-face
{font-family:Wingdings;
panose-1:5 0 0 0 0 0 0 0 0 0;}
@font-face
{font-family:Wingdings;
panose-1:5 0 0 0 0 0 0 0 0 0;}
@font-face
{font-family:Calibri;
panose-1:2 15 5 2 2 2 4 3 2 4;}
@font-face
{font-family:Tahoma;
panose-1:2 11 6 4 3 5 4 4 2 4;}
@font-face
{font-family:Verdana;
panose-1:2 11 6 4 3 5 4 4 2 4;}
/* Style Definitions */
p.MsoNormal, li.MsoNormal, div.MsoNormal
{margin:0in;
margin-bottom:.0001pt;
font-size:11.0pt;
font-family:"Calibri","sans-serif";}
a:link, span.MsoHyperlink
{mso-style-priority:99;
color:blue;
text-decoration:underline;}
a:visited, span.MsoHyperlinkFollowed
{mso-style-priority:99;
color:purple;
text-decoration:underline;}
p.MsoAcetate, li.MsoAcetate, div.MsoAcetate
{mso-style-priority:99;
mso-style-link:"Balloon Text Char";
margin:0in;
margin-bottom:.0001pt;
font-size:8.0pt;
font-family:"Tahoma","sans-serif";}
p.MsoListParagraph, li.MsoListParagraph, div.MsoListParagraph
{mso-style-priority:34;
margin-top:0in;
margin-right:0in;
margin-bottom:0in;
margin-left:.5in;
margin-bottom:.0001pt;
font-size:11.0pt;
font-family:"Calibri","sans-serif";}
span.BalloonTextChar
{mso-style-name:"Balloon Text Char";
mso-style-priority:99;
mso-style-link:"Balloon Text";
font-family:"Tahoma","sans-serif";}
span.EmailStyle20
{mso-style-type:personal;
font-family:"Courier New";
color:windowtext;}
span.EmailStyle21
{mso-style-type:personal;
font-family:"Verdana","sans-serif";
color:#3F31F7;
font-weight:normal;
font-style:normal;}
span.EmailStyle22
{mso-style-type:personal-reply;
font-family:"Courier New";
color:blue;}
.MsoChpDefault
{mso-style-type:export-only;
font-size:10.0pt;}
@page WordSection1
{size:8.5in 11.0in;
margin:1.0in 1.0in 1.0in 1.0in;}
div.WordSection1
{page:WordSection1;}
/* List Definitions */
@list l0
{mso-list-id:1181159027;
mso-list-type:hybrid;
mso-list-template-ids:872440610 1789947978 134807555 134807557 134807553 134807555 134807557 134807553 134807555 134807557;}
@list l0:level1
{mso-level-start-at:0;
mso-level-number-format:bullet;
mso-level-text:-;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;
font-family:"Verdana","sans-serif";
mso-fareast-font-family:Calibri;
mso-bidi-font-family:"Times New Roman";}
@list l0:level2
{mso-level-number-format:bullet;
mso-level-text:o;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;
font-family:"Courier New";}
@list l0:level3
{mso-level-number-format:bullet;
mso-level-text:\F0A7;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;
font-family:Wingdings;}
@list l0:level4
{mso-level-number-format:bullet;
mso-level-text:\F0B7;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;
font-family:Symbol;}
@list l0:level5
{mso-level-number-format:bullet;
mso-level-text:o;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;
font-family:"Courier New";}
@list l0:level6
{mso-level-number-format:bullet;
mso-level-text:\F0A7;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;
font-family:Wingdings;}
@list l0:level7
{mso-level-number-format:bullet;
mso-level-text:\F0B7;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;
font-family:Symbol;}
@list l0:level8
{mso-level-number-format:bullet;
mso-level-text:o;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;
font-family:"Courier New";}
@list l0:level9
{mso-level-number-format:bullet;
mso-level-text:\F0A7;
mso-level-tab-stop:none;
mso-level-number-position:left;
text-indent:-.25in;
font-family:Wingdings;}
ol
{margin-bottom:0in;}
ul
{margin-bottom:0in;}
--></style><!--[if gte mso 9]><xml>
<o:shapedefaults v:ext="edit" spidmax="1026" />
</xml><![endif]--><!--[if gte mso 9]><xml>
<o:shapelayout v:ext="edit">
<o:idmap v:ext="edit" data="1" />
</o:shapelayout></xml><![endif]--></head><body lang=EN-US link=blue vlink=purple><div class=WordSection1><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>Dear Holger,<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>Thank you for your detailed comments. Our experience with degassing the sample is consistent with yours: does not appear to make much of a difference. We have not tried removing the syringe during measurement – Anton Paar rep recommended that we leave it in the plug during the sample measurement. We will try removing the syringe and see if that helps.<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>What concentration range to you normally use for your measurments?<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>Thank you for taking the time to consider my question.<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>Best regards,<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'>John Sumida.<o:p></o:p></span></p><p class=MsoNormal><span style='font-family:"Courier New";color:blue'><o:p> </o:p></span></p><div><div style='border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'>From:</span></b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'> RASMB [mailto:rasmb-bounces@list.rasmb.org] <b>On Behalf Of </b>HGSR (Holger Martin Strauss)<br><b>Sent:</b> Tuesday, September 30, 2014 1:29 AM<br><b>To:</b> John Sumida; rasmb@list.rasmb.org<br><b>Subject:</b> Re: [RASMB] Reproducibility in densitometry measurement<o:p></o:p></span></p></div></div><p class=MsoNormal><o:p> </o:p></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Dear John,<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><o:p> </o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Your observations sound very familiar to our experiences with the DMA5000 and I would love to also hear from other users. Some comments from our experiences:<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><o:p> </o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>It is an extremely sensitive instrument requiring a corresponding experimental standard (which is higher than with a 4000 or 4500)<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Make sure that the calibration is <i>exactly</i> as the measurement. For example, don’t leave the syringe in the plug under the measurement/calibration. It will affect the resonator, at the 5<sup>th</sup> and 6<sup>th</sup> decimal place. Likewise with the plugs or tubing at the other end. We fill our samples, and then remove every tubing, plug, etc. That’s also how we calibrate, but others might have found a better way.<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Gas bubbles can be a problem. Make sure that the temperature of the samples and solvent is similar to your measurement temperature before you dilute and fill them. Likewise, degass preferably at the measurement temperature. We use the small vacuum pump from our ITC-instrument, but in our hands, degassing did not make a huge difference. You can also degass the solvent before you dilute the protein, which can be done more thoroughly than with a protein in it. Any other experiences out there?<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>0.1-0.3 mg/mL is a rather low concentration and small changes in the concentration due to adsorption of your sample to surfaces (from syringes, etc) will have a relatively large effect. But there might be a reason for the rather low concentration.<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Do you determine the density increment or the vbar? A single solution/solvent pair is sufficient for vbar and the highest concentration usually is the most precise measurement. You can of course increase the precision of your parameter by repeated measurements.<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>I’m not aware that a particular salt or concentration can have an effect – but this is more testimony to my ignorance (and bliss, for that matter).<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Accuracy of the concentration measurements is a huge issue and my impression is that the DMA5000’s precision is better than the accuracy of most concentration measurements. Are you using interference optics for the concentration determination? Are you sure that the additional sample handling steps do not change your concentration? Can you run N-determination or AA-analysis in parallel (though they also have their issues)?<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Finally, instruments from a certain period have a CPU-board that will give out after a certain time (in our case, after the warranty expired). I seem to be remember that our measurements were less stable in the weeks before the instrument became unusuable, but I have no documentation to this end. Check with your local AntonPaar subsidiary.<o:p></o:p></span></p><p class=MsoListParagraph style='text-indent:-.25in;mso-list:l0 level1 lfo2'><![if !supportLists]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><span style='mso-list:Ignore'>-<span style='font:7.0pt "Times New Roman"'> </span></span></span><![endif]><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><o:p> </o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>There is of course the experimental alternative of using 18OH2, which avoids the issue of concentration accuracy. But you will still need accuracy in your density and s-values. Moreover, defining the solution conditions wrt species concentrations becomes more of an issue.<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><o:p> </o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'>Cheers, Holger<o:p></o:p></span></p><p class=MsoNormal><span lang=EN-GB style='font-size:10.0pt;font-family:"Verdana","sans-serif";color:#3F31F7'><o:p> </o:p></span></p><div><div style='border:none;border-top:solid #B5C4DF 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'>From:</span></b><span style='font-size:10.0pt;font-family:"Tahoma","sans-serif"'> RASMB [<a href="mailto:rasmb-bounces@list.rasmb.org">mailto:rasmb-bounces@list.rasmb.org</a>] <b>On Behalf Of </b>John Sumida<br><b>Sent:</b> 30 September 2014 00:50<br><b>To:</b> <a href="mailto:rasmb@rasmb.org">rasmb@rasmb.org</a><br><b>Subject:</b> [RASMB] Reproducibility in densitometry measurement<o:p></o:p></span></p></div></div><p class=MsoNormal><span lang=EN-GB><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>Dear RASMB,<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>We are trying to perform a measurement of v-bar using an Anton Paar DMA5000. The measurement is of a glycosylated protein. The densitometer was calibrated against a pure dI water standard and we’ve resorted to air checks between measurements to control for our cleaning protocol: 3 x detergent wash, 3 x dI water rinse, 3 x ethanol rinse, and air drying for 4 minutes or until the reading on the densitometer is 0.0012 g/cm3.<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>We determined the extinction coefficient by synthetic boundary and believe we have accurately measured the concentration of our samples. Stock sample was dialyzed for a little over 48 hours against PBS and dilutions of the stock into dialysate were made in order to generate four solutions ranging from 0.1 to 0.3 mgs/mL.<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>We are getting a values ranging between ~0.60 – 0.70, and are observing that the density measurements are not reproducible beyond the fourth decimal place. <o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>The reason for the lack of reproducibility appears to be that over time, while the sample is in the U-tube, small gas bubbles are observed inside the tube, either due to auto-hydrolysis of the solution or due to the small vibration of the tube or something else I am not aware of. We do not observe this for water alone. Samples were degassed, each for 10 minutes.<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>My questions: <o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>Has anyone observed similar issues when performing this measurement?<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>Is there an optimal buffer system for this measurement – perhaps at a salt concentration less than 100mM?<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>Is there, perhaps, a subtlety in this measurement that I need to be aware of?<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>Thank you for any and all comments returned. They are greatly appreciated.<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><p class=MsoNormal><span style='color:black'>Best regards<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>John Sumida<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>University of Washington</span><span style='font-size:12.0pt;font-family:"Courier New"'><o:p></o:p></span></p><p class=MsoNormal style='text-autospace:none'><span style='color:black'><o:p> </o:p></span></p></div></body></html>