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further washes with equilibration buffer without triton
(2 × 150 μL buffer and 5 min incubations).- Perform a final wash with deionized water (1 × 150 μL and
2 min incubation). - Remove the chips from the reaction chamber and allow the
spots to dry. Add sinapic acid matrix by twice applying 0.7 μL
to each spot (see Note 4). - Equilibrate the chips with 100 mM copper sulfate or 100 mM
nickel sulfate (50 μL and 10 min incubation) and then wash
with deionized water (2 × 150 μL water and 2 min
incubations). - Wash the copper-treated chips further with 50 mM sodium
acetate and the nickel-treated chips with deionized water
(1 × 150 μL and a 5 min incubation). - Further processing is performed as described in the CM10 and
Q10 chip protocol steps 2– 5. - Wash the individual chips three times in methanol (1–2 min
incubations in a suitable container with gentle mixing) and
allowed to dry for at least 1 h, before being placed in the reac-
tion housing. - Equilibrate the chips using the appropriate equilibration buffer
(2 × 150 μL buffer and 10 min incubations). - Add samples to the required final dilution (see Note 3) in
100 μL of buffer and incubate the chips for 30 min. - Wash the chips with equilibration buffer (3 × 150 μL and
5 min incubations) and finally with deionized water (1 × 150 μL
and 2 min incubation). - Remove the chips from the reaction chamber and allow the
spots to dry. Add sinapic acid matrix by twice applying 0.7 μL
to each spot (see Note 4).
In this protocol, samples were spotted directly onto the chip sur-
face, not using a reaction housing, allowing testing of much smaller
sample volumes. This protocol was used for analysis of samples
during purification of peaks by fractionation and passive elution
from gels.
For NP20 chips:- Pre-wet the spots with deionized water, using an on/off pipet-
ting technique. - Add samples onto the spots (2 × 1 μL additions) and incubate
the chips in a humidity chamber for 30 min with gentle
shaking.
3.1.2 IMAC Chip Protocol
3.1.3 H50 Chip Protocol
3.1.4 Spot Protocol
for NP20 and H50 Chips
Peter Kelly