ProteOn XPR36, Руководство пользователя

Страница: 82 / 288

ProteOn XPR36 | Experiments
64
Experiment Preparation
Dissolving and Aliquoting ProteOn EDAC
and Sulfo-NHS
Use the ProteOn amine coupling kit to couple ligands to ProteOn G-series
sensor chips that have Bio-Rad proprietary chip surface chemistry. The highly
soluble sulfo-NHS (N-hydroxysuccinimide), in conjunction with EDAC (1-ethyl-
3-[3-dimethylaminopropyl] carbodiimide HCl), efficiently activates the chip’s
surface carboxyl groups and immobilizes ligand proteins to the chip surface
without multiple crosslinks. After injection of the ligand, the chip surface is
deactivated with ethanolamine. The ethanolamine step blocks any remaining
activated carboxyl groups and neutralizes their residual surface charges,
which in turn reduces nonspecific binding and electrostatic interference with
analyte binding.
To dissolve and aliquot:
1. Store EDAC and sulfo-NHS powder at –20°C to –80°C before use.
2. Add 75 ml cold deionized or distilled water to ProteOn EDAC (0.575 g)
to dissolve it and rinse it out of its bottle. The final concentration will be
40 mM. Divide it into aliquots based on your usage, typically 0.5 to 1 ml
each.
3. Add 75 ml cold deionized or distilled water to ProteOn sulfo-NHS (0.163
g) to dissolve it and rinse it out of its bottle. The final concentration will
be 10 mM. Divide it into aliquots based on your usage, typically 0.5 to 1
ml each.
4. For moderate activations, thaw and mix aliquots of EDAC and sulfo-
NHS in a 1:1 ratio without dilution immediately prior to injection. Inject
100 to 150 ul at 30 ul/min. Discard any unused EDAC and sulfo-NHS;
do not refreeze them.
Note: Separate EDAC and sulfo-NHS solutions can be stored at
4–8 °C for several hours. Once mixed, the solution must be used
immediately.
5. To increase or decrease the amount of ligand being immobilized on the
chip surface, (1) increase or decrease the ligand concentration; for
example, doubling the ligand concentration will approximately double
the amount of ligand bound; (2) increase or decrease the contact time;
or (3) decrease the EDAC used in the activation step (increasing the
EDAC above 40 mM will not significantly increase ligand immobilization