IMAC30 Chip Pretreatment
Assemble the 96-well bioprocessor for chip pretreatment by placing 12 strips of protein chips in the base clamp assembly and then putting a 96-hole rubber gasket sheet and a 96-well bioprocessor reservoir on top of the chips.
Clamp this sandwich tight with the 96 incubation wells in the reservoir placed directly on top of the 96 chip spots.
Add 50 µL of 100 mM CuSO4 solution into each well in the reservoir making direct contact with each IMAC30 chip spot.
Get rid of air bubbles if present.
Shake the chips in the bioprocessor for 5 min at the same speed and time as before in the orbital shaker at RT to allow copper ions from CuSO4 solution to bind to the chip surface.
Pipet away the CuSO4 solution.
Rinse the chip spot in each well with 100 µL of Milli-Q grade of water and shake again under the same condition for 1 min at RT.
Rinse each well with 100 µL of sodium acetate buffer at pH 4.0 (IMAC30 chip neutralizing buffer) for 5 min with shaking at RT.
Remove the neutralizing buffer after shaking.
Rinse the well again with 100 µL of Milli-Q water by shaking for 1 min at room temperature.
Add 150 µL of 100 mMsodium phosphate buffer containing 0.5 mol/L NaCl (IMAC30 chip binding buffer) onto each well and shake for 5 min at RT to wash the chip spot.
Remove the buffer after shaking.
Wash with the same IMAC30 chip-binding buffer one more time.
Remove the buffer after washing.
Proceed immediately to Subheading 3.6.1. for sample binding without letting the chip surface to dry.