Bio-Rad ProteinChip Serum Fractionation Kits User Manual

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3. With an 8-channel pipet, add 200 µl of rehydration buffer to

each well.

4. Carefully seal the plates with microplate sealing strips and

shake by hand for a few minutes (some liquid may come
through the membrane) until the sorbent appears to be
resuspended in solution.

5. Mix the filtration plate on the MicroMix 5 (form 48, amp 7) for

60 minutes at RT. Carefully remove the sealing strips. Place
plate on vacuum collar and then place the vacuum collar and
plate on vacuum manifold.

Note: It is extremely important to mix the sorbent and rehydration buffer well to avoid
plugging wells during fractionation. Check visually that mixing is adequate. If necessary,
adjust the MicroMix form and amp settings. Mixing should be vigorous enough to
ensure that all sorbent is in contact with buffer without the mixture reaching the top of
the well. If mixing is still not adequate after changing the MicroMix settings, it may be
necessary to adjust settings in the MicroMix 5 software (see Appendix B).

Step 2: Sample Preparation

1. Bring serum samples to ambient temperature. Spin at

20,000 g for 10 minutes at 2–8°C.

2. Aliquot 20 µl of serum sample to each well of a standard

V-bottom 96-well microplate.

3. Add 30 µl of ProteinChip U9 buffer to each well.

4. Cover microplate with adhesive sealing film and mix on the

MicroMix 5 (set at 20, 5, 20) for 20 minutes at 2–8°C.

Step 3: Preparation of U1 Buffer

1. Add 10 ml of ProteinChip U9 buffer solution to 80 ml of

rehydration buffer (50 mM Tris-HCl) to produce U1 buffer
(1 M urea, 0.2% CHAPS, 50 mM Tris-HCl, pH9). The volume is
enough for 1 complete plate. If you are using part of the plate,
adjust volume accordingly.

Detailed Use Protocol

The following protocol can be used with the Biomek 2000 or 3000
laboratory automation workstation or can be performed manually
using a vacuum manifold. We recommend a vacuum setting of
15 in Hg.

Notes:
1. When using the filtration plate in the MicroMix 5, make sure that the plate is securely

held in the manifold and that the bottom of the plate is not touching the surface of
the mixer.

2. The settings recommended for the MicroMix 5 may vary from mixer to mixer. If you

experience problems with mixing, you may need to adjust the settings. See
Appendix B for instructions.

3. When removing the foil seal from the ProteinChip Q filtration plate, you may notice

some of the sorbent adhered to the foil seal. The amount should be relatively
uniform across all wells. This is normal and has not been found to adversely affect
the performance of the kit.

Step 1: Q Ceramic HyperD F Sorbent Rehydration

The filtration plate should be used directly after rehydration.

1. Tap the filtration plate on the workbench several times to make

sure that all of the dry Q ceramic HyperD F sorbent is settled to
the bottom of the plate.

2. Take the filtration plate out of the pouch and carefully remove the

top seal on the filtration plate.

Note: If using only part of the filtration plate, with a sharp blade remove only enough of
the foil seal to reveal the columns needed. Alternatively, remove the whole seal, and
reseal the unneeded wells with the microplate strips provided. While processing
samples, only the wells in use should be uncovered. After using part of the plate,
reseal the used wells with microplate strips and mark those columns as used.