Elution of Serum Fractions
After shaking, place a 96-well F1 collection plate under the filter plate and centrifuge at 1600g for 1 min to collect the flow-through fraction in the wells of the F1 collection plate.
Add 100 µL of E1 elution buffer (at pH 9.0) to each well in the filter plate containing the serum bound beads and shake vigorously again on orbital shaker (speed and time as before) for 10 min at RT.
Collect the eluate in the same collection plate F1 by centrifugation at 1600g for 1 min again.
This represents fraction 1, which contains both the flow-through fraction at pH 9.0 and the pH 9.0 eluate.
Add 100 µL of E2 elution buffer (at pH 7.0) to each well in the filter plate and again shake at the same speed and time.
Collect the eluate in another new F2 collection plate by centrifugation as before.
Add 100 µL of E2 elution buffer again to each filter well and elute a second time by shaking and centrifugation as before onto F2 collection plate again.
This represents fraction 2, which contains the pH 7.0 eluate.
Elute the serum protein bound beads similarly in turn with E3, E4, and E5 elution buffers as above resulting in fractions 3, 4, and 5 containing pH 5.0, pH 4.0, and pH 3.0 eluates, respectively, in three new F3, F4, and F5 collection plates.
Final elution is achieved by adding E6 organic solvent elution buffer and centrifugation at 2000g for 5 min giving rise to fraction 6 containing the organic solvent eluate in collection plate F6.
Freeze fractions 1–6 in collection plates E1–6 at −70°C until the chip binding protocol proceeds.