ProteOn XPR36, User Manual

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ProteOn XPR36 System | ProteOn System Troubleshooting
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Nonspecific Binding
Nonspecific binding, or NSB, is defined as the binding of an analyte or other
component(s) in the sample or running buffer directly to the sensor chip
surface and not to the target ligand. NSB is characterized by significant
binding response signals on the reference spots. These events have the
potential to skew assay results. A typical symptom of NSB might be a bad fit
of the experimental data during kinetic analysis.
B
ACKGROUND
The most common cause of NSB is electrostatic attraction of a positively
charged analyte or other component to the negatively charged binding layer.
This is called “Electrostatic NSB.”
Other sources of NSB include chemical interactions of the analyte or other
components with the binding layer, such as hydrophobic interactions,
hydrogen bonding, or binding to nanoscopic areas of exposed gold on the
surface. These are called “Non-electrostatic NSB.”
Compounds with a propensity for NSB include:
• Positively charged proteins: pl > pH of the running buffer
• Compounds containing thiol groups or related forms of sulfur
known to show high affinity to gold, such as cysteine-containing
peptides
• Molecules that naturally bind polysaccharides might exhibit NSB to
the alginate-containing binding layer, such as lectins
• Biological solutions such as serum or crude lysates, supernatants
Note: In many cases, it can be difficult to identify the type of
NSB. Furthermore, it is possible that more than one type is
causing the problem. Knowing the properties of an analyte can
help assess the NSB source.
The Quality Control procedures used in the manufacture of ProteOn sensor
chips contain a standard test for NSB. However, certain molecules, such as
those described above, may still cause NSB. This is because NSB can also
come from inadequate cleaning of the ProteOn fluidics, running an
inappropriate buffer, and a number of other causes.