Ion Exchange Fractionation of Serum Proteins
Owing to the protein complexity in the serum sample, an initial fractionation in Q Ceramic Hyper D F ion exchange sorbent beads is performed to generate serum fractions for chip binding experiments (Fig. 1) (10,11).

Washing of Ion Exchange Beads
Wash Q Ceramic Hyper D Fanion exchange sorbent beads three times each with five bed volumes of W1 bead washing buffer.
Drain the washing buffer each time in vacuum after washing.
Keep beads in 50% suspension in W1 buffer at RT.

Denaturation of Serum Proteins
Thaw serum from −70°C freezer immediately before serum fractionation.
Aliquot 20 µL of serum to each well in a 96-well sample plate.
Add 30 µL of D1 denaturing buffer to the well containing the serum to denature the serum proteins.
Shake the sample plate on a Micromix 5-01 shaker vigorously (in an amplitude of 7 and form of 20 Hg) for 20 min at 4°C to mix the serum with the D1 buffer well.

Equalibration of Ion Exchange Beads and Binding of Serum Samples
Add 180 µL of 50% suspension of ion exchange beads to each well of a 96-well Silent Screen filter plate and drain off the W1 bead-washing buffer by vacuum.
Wash the ion exchange beads in each well three more times by adding 200 µL of Q1 bead equilibration buffer to the beads and draining off the buffer by vacuum.
After the last vacuum drain, transfer 50 µL of the denatured serum sample from the well in the sample plate to the corresponding well in the filter plate containing the equilibrated ion exchange beads.
Add 50 µL of Q1 buffer to the well in the sample plate where serum was originally placed and rinse the residual serum by pipetting up and down five times.
Transfer the 50 µL of the serum rinse to the well in the filter plate containing the mixture of denatured serum sample and ion exchange beads.
Shake the filter plate vigorously on the Micromix shaker (in an amplitude of 7 and form of 20 Hg) for 30 min at 4°C to mix the serum and beads well.

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.