TECHNICAL
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Discussion
In summary, the effect of immobilization buffers on
different types of proteins typically analyzed in SPR
assays is quickly and easily assessed using the Tycho
NT.6. Conditions that can impact the folded state of
the protein can be identified with the system, which
provides insight into SPR assay development and
testing. Experimental runs on the Tycho NT.6 take
three minutes, requires minimal sample handling
and use less than 10 µL of sample material. This
translates into obtaining results faster (~10x) than
using conventional pH scouting experiments. Tycho
NT.6 uses single-use capillaries to run experiments
and affords significant cost savings (~60-fold less)
compared to a standard biosensor chip.
Methodology
Measurement
Time
(minutes)
Protein
Required
Per Test
Experimental
Cost
Tycho NT.6 3 0.5-1 µg 2€ / 2.40USD
SPR* 38 5-10 µg 125€ /
147USD
*Biacore CM5-Chip (standard)
Table 1. Time, sample and cost-savings using the Tycho NT.6 over standard SPR
assay development.
The Tycho NT.6 provides significant time and cost
savings as well as using very low amounts of material
to enable researchers to make better decisions in
optimizing biosensor assay development and testing.
Figure 2 Quick evaluation of immobilization buffer conditions with Tycho NT.6. Two proteins were tested in standard SPR immobilization buffers and in
PBS as a reference. A kinase (le ) is fully denatured at pH values 4.0 and 4.5, indicated by the high initial fluorescence ratio and the absence of an unfolding
event. The protein is partially denatured at pH 5.0, indicated by the increased initial ratio and the shi ed unfolding event when compared to the reference
sample. The best option for an immobilization buffer to be tested in SPR which guarantees the structural integrity of the kinase is the buffer with pH 5.5. In
contrast, a mAb (right) is stable in all tested immobilization buffers.
