ProteOn XPR36, Руководство пользователя

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Nonspecific Binding
173
A
DDRESSING THE
C
AUSES OF
NSB
There are a number of methods to prevent NSB. These are specific to the
nature of each NSB and listed in order of importance.
• Optimize the appropriate running and sample buffer for your
application (see the effects of working with positively charged
proteins, above)
• When using complex biological samples like serum or crude
lysates, dilute the sample as much as possible with running buffer;
a 5–10 fold dilution is recommended
• Purify the analyte or sample, when possible
• Try different immobilization chemistries. For example, use a
biotinylated ligand on an NLC chip
• Immobilize an analyte that has NSB as the ligand, if possible
• Immobilize a non-relevant protein to the reference channel, like
BSA
• Increase salt concentration in the sample and running buffers to
reduce electrostatic interactions
• Elevate the pH of the analyte and running buffers to reduce positive
charge contributing to electrostatic NSB
• Perform intense activation/de-activation steps on active and
reference channels before ligand immobilization. The negative
charge in these channels will be reduced, and electrostatic NSB
issues may be resolved. However, bear in mind that a significant
decrease in binding capacity will result.
Note: This action must be performed before ligand
immobilization due to possible chemical damage to an
immobilized ligand.
• Add or increase the concentration of detergents like Tween 20 in
the analyte sample and running buffer to reduce hydrophobic or
non-electrostatic NSB
• Re-test your binding assay with a fresh chip, preferably from a
different lot. Doing so will eliminate chip-specific defects as a
cause