[RASMB] SPR question

[Huber, Sylwia]

Dear John,

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.

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.

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.

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.

Kind regards,
Sylwia

Mit freundlichen Grüssen/With Kind Regards



*Dr. Sylwia Huber*
Principal Scientist, *Biophysics Team Head*
Roche Innovation Center Basel
F. Hoffmann-La Roche Ltd., Chemical Biology
Building 65/203, Grenzacherstrasse 124
CH-4070 Basel, Switzerland
Phone: +41/61/6879507


On Mon, Dec 11, 2017 at 8:40 PM, John Sumida <jpsumida at uw.edu> wrote:

> Dear RASMB,
>
>
>
> This question, is in relation to a Biacore T200 instrument and an artifact
> we are seeing at high sample injection concentrations (20uM to 1mM).
>
>
>
> 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.
>
>
>
> The observables are
>
> 1.       Regardless of the duration of the injection, the signal response
> rises to a maximum halfway through the injection and then decreases
>
> 2.       This behavior appears to be concentration dependent.
>
> 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.
>
>
>
> 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.
>
>
>
> These types of concentrations regimes and exposure times are not the
> injection protocols I normally use, thus I have not seen this behavior
> previously.
>
>
>
> *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?*
>
>
>
> I would like to thank the list in advance for any comments or advice you
> may provide, and wish everyone a happy holiday season.
>
>
>
> Best regards,
>
> John Sumida
>
> Molecular Analysis Facility
>
> University of Washington
>
>
>
> _______________________________________________
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> RASMB at list.rasmb.org
> http://list.rasmb.org/listinfo.cgi/rasmb-rasmb.org
>
>
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