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<p>Hi-</p>
<p>I am not an SPR expert, so this may be a naive question on my part. Are you dialyzing your samples against the solvent prior to running them? The idea here is that you may be observing an refractive index artifact at the midpoint injection time. Again, just
a thought.<br>
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<p>Best wishes,</p>
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<div style="font-family:Tahoma; font-size:13px"><font size="3"><font face="Times New Roman">Tom<br>
University of New Hampshire<br>
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<div id="divRplyFwdMsg" dir="ltr"><font style="font-size:11pt" face="Calibri, sans-serif" color="#000000"><b>From:</b> RASMB <rasmb-bounces@list.rasmb.org> on behalf of Huber, Sylwia <sylwia.huber@roche.com><br>
<b>Sent:</b> Wednesday, December 13, 2017 7:18 AM<br>
<b>To:</b> John Sumida<br>
<b>Cc:</b> RASMB<br>
<b>Subject:</b> Re: [RASMB] SPR question</font>
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<div dir="ltr">Dear John,
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<div>such deviation in the association phase we have observed in our laboratory as well. We did comparison study with the same protein-sample system on the Biacore 3000 and found that the deviation you observe are typical for the T200 instrument. </div>
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<div>They occur only for some proteins, in our cases, very often for membrane proteins, or systems where you have to work with detergents or exotic buffers.</div>
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<div>We found the same data as you. The deviation is independent on the injection time, it occurs almost always around the middle of the association phase. We found that this happens also for the buffer injection on the protein but not on the reference (channel).
We found also that it is dependent on the flow rate. The highest flow rate the strongest the deviation.</div>
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<div>I had a lot of discussions with Biacore Specialists from GE, but up to now we did not find the reason for this observation. We speculated different things, e.g. pressure lowering during the injection time, but up to now we have still no answer on that.
If anybody will find the solution of this problem, please let us know. I am also very curious about this.<br>
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<div>Kind regards,</div>
<div>Sylwia</div>
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<div dir="ltr"><font size="1">Mit freundlichen Grüssen/With Kind Regards</font></div>
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<b><br>
</b><b>Dr. Sylwia Huber</b><br>
Principal Scientist, <i>Biophysics Team Head</i></font></div>
<div dir="ltr"><font size="1">Roche Innovation Center Basel</font>
<div><font size="1">F. Hoffmann-La Roche Ltd., Chemical Bi</font><span style="font-size:x-small">ology</span></div>
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<div><span style="font-size:x-small">Building 65/203, </span><span style="font-size:x-small">Grenzacherstrasse 124</span></div>
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Phone: +41/61/6879507</font>
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<div class="gmail_quote">On Mon, Dec 11, 2017 at 8:40 PM, John Sumida <span dir="ltr">
<<a href="mailto:jpsumida@uw.edu" target="_blank">jpsumida@uw.edu</a>></span> wrote:<br>
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<p class="MsoNormal" style="text-autospace:none"><span style="color:black">Dear RASMB,<u></u><u></u></span></p>
<p class="MsoNormal" style="text-autospace:none"><span style="color:black"><u></u> <u></u></span></p>
<p class="MsoNormal" style="text-autospace:none"><span style="color:black">This question, is in relation to a Biacore T200 instrument and an artifact we are seeing at high sample injection concentrations (20uM to 1mM).
<u></u><u></u></span></p>
<p class="MsoNormal" style="text-autospace:none"><span style="color:black"><u></u> <u></u></span></p>
<p class="MsoNormal" style="text-autospace:none"><span style="color:black">In discussing these observations, it has been suggested that these observations are a function of the T200 microfluidics which exhibit a reversal of sample/analyte flow at high sample
concentrations. </span><span style="color:black"><u></u><u></u></span></p>
<p class="MsoNormal" style="text-autospace:none"><span style="color:black"><u></u> <u></u></span></p>
<p class="MsoNormal" style="text-autospace:none"><span style="color:black">The observables are<u></u><u></u></span></p>
<p class="m_132486770515307473MsoListParagraph" style="text-autospace:none"><u></u><span style="color:black"><span>1.<span style="font:7.0pt "Times New Roman"">
</span></span></span><u></u><span style="color:black">Regardless of the duration of the injection, the signal response rises to a maximum halfway through the injection and then decreases
<br>
<img id="m_132486770515307473Picture_x0020_3" width="544" height="158" src="cid:image007.jpg@01D37274.D3030680"><u></u><u></u></span></p>
<p class="m_132486770515307473MsoListParagraph" style="text-autospace:none"><u></u><span style="color:black"><span>2.<span style="font:7.0pt "Times New Roman"">
</span></span></span><u></u><span style="color:black">This behavior appears to be concentration dependent.<br>
<img id="m_132486770515307473Picture_x0020_4" width="346" height="268" src="cid:image008.jpg@01D37274.D3030680"><u></u><u></u></span></p>
<p class="MsoNormal">It’s been suggested that this is a sample dependent phenomenon, where a reversal of flow occurs in the microfluidic channel above the sensor if the sample concentration exceeds some value that is a function of the molecule size and its
diffusivity. <u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">The effect I am seeing appears to be concentration dependent, and the response above the sensor surface is unusual in that above a certain concentration, the response reaches a maximum value almost exactly halfway through the injection
time/volume (63% in both examples above) regardless of the injection time. It makes sense that at long injection times there would be a decrease in the response during the end of the injection time due to dispersion of the sample plume in the running buffer,
but in this particular case, while this response is observed at 240 seconds, a relatively long time to use on the Biacore instrument, it is also observed at 120sec which I would not consider a long injection time.<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal">These types of concentrations regimes and exposure times are not the injection protocols I normally use, thus I have not seen this behavior previously.
<u></u><u></u></p>
<p class="MsoNormal"><u></u> <u></u></p>
<p class="MsoNormal"><b>In your estimation, is it possible for flow to be reversed within a microfluidic channel at high sample concentration and for this to result in the observed behavior?<u></u><u></u></b></p>
<p class="MsoNormal"><u></u> <u></u></p>
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<p class="MsoNormal"><span style="color:black">I would like to thank the list in advance for any comments or advice you may provide, and wish everyone a happy holiday season.<u></u><u></u></span></p>
<p class="MsoNormal"><span style="color:black"><u></u> <u></u></span></p>
<p class="MsoNormal"><span style="color:black">Best regards,<u></u><u></u></span></p>
<p class="MsoNormal"><span style="color:black"><img id="m_132486770515307473Picture_x0020_2" width="119" height="48" src="cid:image009.jpg@01D37274.D3030680"><u></u><u></u></span></p>
<p class="MsoNormal"><span style="color:black">John Sumida<u></u><u></u></span></p>
<p class="MsoNormal"><span style="color:black">Molecular Analysis Facility<u></u><u></u></span></p>
<p class="MsoNormal"><span style="color:black">University of Washington<u></u><u></u></span></p>
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<p class="MsoNormal"><u></u> <u></u></p>
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