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</o:shapelayout></xml><![endif]--></head><body bgcolor=white lang=EN-US link=blue vlink=purple><div class=WordSection1><p class=MsoNormal><span style='color:black'>Thank you all for your responses on and off list. I have attached a jpg file in this email that illustrates the artifact we are observing.<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'>I do have additional information that may be useful.<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'>To answer Tom’s question, yes the sample prep in this current case included an exhaustive dialysis against running buffer (10mM HEPES, 150mM NaCl, 3mM MgCl2, 0.05%P20, pH=7.4) – we’ve tried this experiment a number of times with the same result and had been focused on incomplete dialysis of the sample being the problem, so we were overly cautious in this regard for the last experiment. Additionally, samples were microfuged and pre-filtered with a 0.22um syringe filter; additionally, there is no known propensity of this protein to self-associate at these concentrations though this has not been determined by this lab.<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 term “flow reversal,” used in my initial post, is a term that was used to describe the process to me, and it is inaccurate. While there appear to be examples of actual flow reversals in microfluidic channels in the literature under specific conditions, this has not, to my knowledge been reported, for the Biacore microfluidics system. The use of the term refers to the sequences of steps the instrument proceeds through during the injection of sample where sample is initially aspirated into a sample loop and then the flow is “reversed” and injected onto the sensor surface. Similar deviations in surface responses are known to occur in microfluidic systems, in general, where pulsed solution delivery is used and where the direction of flow is reversed in order to inject sample into the channel (ie this would include rigs that utilize sample loops for injection), and one current working theory is that the artifact is associated with this injection format. Instruments, which do not use a sample loop, relying instead on direct injection of the sample, do not, apparently, exhibit this behavior. <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'>As this lab, and it appears now also other labs, have observed this effect for proteins, I need to add that this behavior is usually associated with small hydrophobic molecules. In our case, we are seeing these injection phase deviations both for hydrophobic small molecule drugs as well as for a small to medium sized protein at concentrations (12nM) well below the level I expect to see noticeable changes in refractive index upon mixing. <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'>What is interesting to me, is why this happens for some molecules and not others. I suspect that one possible explanations would be a gradated change in the refractive index of the solution as a result of reversing the direction of flow, perhaps akin to what might happen in a synthetic boundary experiment, but the deviations are quite reproducible and I would not have expected that in the absence of a centrifugal field.<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'>Thanks again for taking the time to consider this topic.<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.<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'>Molecular Analysis Facility.<o:p></o:p></span></p><p class=MsoNormal><span style='color:black'><o:p> </o:p></span></p><div><div style='border:none;border-top:solid #E1E1E1 1.0pt;padding:3.0pt 0in 0in 0in'><p class=MsoNormal><b><span style='font-size:11.0pt;font-family:"Calibri",sans-serif'>From:</span></b><span style='font-size:11.0pt;font-family:"Calibri",sans-serif'> RASMB [mailto:rasmb-bounces@list.rasmb.org] <b>On Behalf Of </b>Laue, Thomas<br><b>Sent:</b> Wednesday, December 13, 2017 8:14 AM<br><b>To:</b> Huber, Sylwia <sylwia.huber@roche.com>; John Sumida <jpsumida@uw.edu><br><b>Cc:</b> RASMB <rasmb@list.rasmb.org><br><b>Subject:</b> Re: [RASMB] SPR question<o:p></o:p></span></p></div></div><p class=MsoNormal><o:p> </o:p></p><p><span style='color:black'>Hi-<o:p></o:p></span></p><p><span style='color:black'>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.<o:p></o:p></span></p><p><span style='color:black'>Best wishes,<o:p></o:p></span></p><div id=Signature><div name=divtagdefaultwrapper><div><p class=MsoNormal style='margin-bottom:12.0pt'><span style='color:black'>Tom<br>University of New Hampshire</span><span style='font-size:10.0pt;font-family:"Tahoma",sans-serif;color:black'><o:p></o:p></span></p></div></div></div><div><div class=MsoNormal align=center style='text-align:center'><span style='color:#212121'><hr size=2 width="98%" align=center></span></div><div id=divRplyFwdMsg><p class=MsoNormal><b><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:black'>From:</span></b><span style='font-size:11.0pt;font-family:"Calibri",sans-serif;color:black'> RASMB <<a href="mailto:rasmb-bounces@list.rasmb.org">rasmb-bounces@list.rasmb.org</a>> on behalf of Huber, Sylwia <<a href="mailto:sylwia.huber@roche.com">sylwia.huber@roche.com</a>><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</span><span style='color:#212121'> <o:p></o:p></span></p><div><p class=MsoNormal><span style='color:#212121'> <o:p></o:p></span></p></div></div><div><div><p class=MsoNormal><span style='color:#212121'>Dear John, <o:p></o:p></span></p><div><p class=MsoNormal><span style='color:#212121'><o:p> </o:p></span></p></div><div><p class=MsoNormal><span style='color:#212121'>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. <o:p></o:p></span></p></div><div><p class=MsoNormal><span style='color:#212121'><o:p> </o:p></span></p></div><div><p class=MsoNormal><span style='color:#212121'>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.<o:p></o:p></span></p></div><div><p class=MsoNormal><span style='color:#212121'><o:p> </o:p></span></p></div><div><p class=MsoNormal><span style='color:#212121'>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.<o:p></o:p></span></p></div><div><p class=MsoNormal><span style='color:#212121'><o:p> </o:p></span></p></div><div><p class=MsoNormal><span style='color:#212121'>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.<o:p></o:p></span></p></div><div><p class=MsoNormal><span style='color:#212121'><o:p> </o:p></span></p></div><div><p class=MsoNormal><span style='color:#212121'>Kind regards,<o:p></o:p></span></p></div><div><p class=MsoNormal><span style='color:#212121'>Sylwia<o:p></o:p></span></p></div></div><div><p class=MsoNormal><span style='color:#212121'><br clear=all><o:p></o:p></span></p><div><div><div><div><div><div><div><div><div><div><div><div><div><div><div><p class=MsoNormal><span style='font-size:7.5pt;color:#212121'>Mit freundlichen Grüssen/With Kind Regards</span><span style='color:#212121'><o:p></o:p></span></p></div><div><p class=MsoNormal><span style='color:#212121'><o:p> </o:p></span></p></div><div><p class=MsoNormal><span style='font-size:7.5pt;color:#212121'><br><b><br>Dr. Sylwia Huber</b><br>Principal Scientist, <i>Biophysics Team Head</i></span><span style='color:#212121'><o:p></o:p></span></p></div><div><p class=MsoNormal><span style='font-size:7.5pt;color:#212121'>Roche Innovation Center Basel</span><span style='color:#212121'> <o:p></o:p></span></p><div><p class=MsoNormal><span style='font-size:7.5pt;color:#212121'>F. Hoffmann-La Roche Ltd., Chemical Bi</span><span style='font-size:10.0pt;color:#212121'>ology</span><span style='color:#212121'><o:p></o:p></span></p></div><div><div><p class=MsoNormal><span style='font-size:10.0pt;color:#212121'>Building 65/203, Grenzacherstrasse 124</span><span style='color:#212121'><o:p></o:p></span></p></div></div><div><p class=MsoNormal><span style='font-size:7.5pt;color:#212121'>CH-4070 Basel, Switzerland<br>Phone: +41/61/6879507</span><span style='color:#212121'> <o:p></o:p></span></p><div><p class=MsoNormal><span style='color:#212121'><o:p> </o:p></span></p></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div></div><p class=MsoNormal><span style='color:#212121'><o:p> </o:p></span></p><div><p class=MsoNormal><span style='color:#212121'>On Mon, Dec 11, 2017 at 8:40 PM, John Sumida <<a href="mailto:jpsumida@uw.edu" target="_blank">jpsumida@uw.edu</a>> wrote:<o:p></o:p></span></p><blockquote style='border:none;border-left:solid #CCCCCC 1.0pt;padding:0in 0in 0in 6.0pt;margin-left:4.8pt;margin-right:0in'><div><div><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;text-autospace:none'><span style='color:black'>Dear RASMB,</span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;text-autospace:none'><span style='color:black'> </span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;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). </span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;text-autospace:none'><span style='color:black'> </span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;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:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;text-autospace:none'><span style='color:black'> </span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto;text-autospace:none'><span style='color:black'>The observables are</span><span style='color:#212121'><o:p></o:p></span></p><p class=m132486770515307473msolistparagraph style='text-autospace:none'><span style='color:black'>1.</span><span style='font-size:7.0pt;color:black'> </span><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 border=0 width=544 height=158 id="m_132486770515307473Picture_x0020_3" src="cid:image001.jpg@01D37407.05BF69E0"></span><span style='color:#212121'><o:p></o:p></span></p><p class=m132486770515307473msolistparagraph style='text-autospace:none'><span style='color:black'>2.</span><span style='font-size:7.0pt;color:black'> </span><span style='color:black'>This behavior appears to be concentration dependent.<br><img border=0 width=346 height=268 id="m_132486770515307473Picture_x0020_4" src="cid:image002.jpg@01D37407.05BF69E0"></span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:#212121'>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. <o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:#212121'> <o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:#212121'>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.<o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:#212121'> <o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:#212121'>These types of concentrations regimes and exposure times are not the injection protocols I normally use, thus I have not seen this behavior previously. <o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:#212121'> <o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><b><span style='color:#212121'>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?</span></b><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:#212121'> <o:p></o:p></span></p><div><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><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.</span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:black'> </span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:black'>Best regards,</span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:black'><img border=0 width=119 height=48 id="m_132486770515307473Picture_x0020_2" src="cid:image003.jpg@01D37407.05BF69E0"></span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:black'>John Sumida</span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:black'>Molecular Analysis Facility</span><span style='color:#212121'><o:p></o:p></span></p><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:black'>University of Washington</span><span style='color:#212121'><o:p></o:p></span></p></div><p class=MsoNormal style='mso-margin-top-alt:auto;mso-margin-bottom-alt:auto'><span style='color:#212121'> <o:p></o:p></span></p></div></div><p class=MsoNormal style='margin-bottom:12.0pt'><span style='color:#212121'><br>_______________________________________________<br>RASMB mailing list<br><a href="mailto:RASMB@list.rasmb.org">RASMB@list.rasmb.org</a><br><a href="https://urldefense.proofpoint.com/v2/url?u=http-3A__list.rasmb.org_listinfo.cgi_rasmb-2Drasmb.org&d=DwMFaQ&c=c6MrceVCY5m5A_KAUkrdoA&r=hAcbVT23dt1uq6AGxObZKg&m=IAvBv4haDMBbLBr7cH1MgDP7yDcnQn0VFscS8hjZHzs&s=X3zW3fWljk290BGdX95fu3s7Z5l3ILTtJNapncH7HD8&e=" target="_blank">http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org</a><o:p></o:p></span></p></blockquote></div><p class=MsoNormal><span style='color:#212121'><o:p> </o:p></span></p></div></div></div></div></body></html>