Bio-Rad ProteinChip Serum Fractionation Kits User Manual

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Step 6: Fraction Collection

Note: To avoid cross-contamination between wells, apply adhesive sealing film on the
microplate during the mixing step. Remove the film before applying a vacuum, and
replace with a new piece for each mixing.

Fraction 1

1. Place the 96-well microplate labeled F1 underneath the

filtration plate.

2. Apply the vacuum and collect the flowthrough into the F1 plate.

3. Add 100 µl of wash buffer 1 to each well of the filtration plate.

4. Mix for 10 minutes on the MicroMix 5 (set at 20, 7, 10) at RT.

5. Apply the vacuum and collect the eluent into the F1 plate.

Note: Fraction 1 contains the flowthrough and the pH 9 eluent.

Fraction 2

1. Add 100 µl of wash buffer 2 to each well of the filtration plate.

2. Mix for 10 minutes on the MicroMix 5 (set at 20, 7, 10) at RT.

3. Place the 96-well microplate labeled F2 underneath the

filtration plate.

4. Apply the vacuum and collect the eluent into the F2 plate.

5. Repeat procedures 1–4.

Note: Fraction 2 contains the pH 7 eluent.

Fraction 3

1. Add 100 µl of wash buffer 3 to each well of the filtration plate.

2. Mix for 10 minutes on the MicroMix 5 (set at 20, 7, 10) at RT.

3. Place the 96-well microplate labeled F3 underneath the

filtration plate.

4. Apply the vacuum and collect the eluent into the F3 plate.

5. Repeat procedures 1–4.

Note: Fraction 3 contains the pH 5 eluent.

Step 4: Equilibration of Q Ceramic HyperD F Sorbent With U1 Buffer

1. After step 1.4, place the waste collection plate underneath the

filtration plate and apply a vacuum to remove the buffer from the
filtration plate.

2. Add 200 µl of rehydration buffer to each well.

3. Apply vacuum to remove the buffer in the filtration plate.

4. Repeat steps 4.2 and 4.3 three times with rehydration buffer and

an additional three times with U1 buffer. Empty the waste plate
under the filtration plate as necessary between washes.

5. The sorbent in the filtration plate is now ready to bind sample.

Step 5: Binding Sample With Sorbent

1. Pipet 50 µl of sample from each well of the sample microplate to

the corresponding well in the 96-well filtration plate.

2. Add 50 µl of U1 buffer to each well of the sample microplate.

3. Mix 5 times.

4. Pipet 50 µl from each well of the sample microplate to the

corresponding well in the 96-well filtration plate.

Note: This step is included because there is a dead volume when pipetting with the
robot. When the robot pipets to collect the sample initially, it does not collect all of the
material. The addition of 50 µl of U1 buffer and mixing allows the residual material to
be collected and added to the first 50 µl.

5. Cover the filtration plate with adhesive sealing film and mix on

the MicroMix 5 (set at 20, 7, 30) for 30 minutes at 2–8°C.