Bio-Rad ProteinChip Serum Fractionation Kits - Manual (Page 3)

© 2006 Bio-Rad Laboratories, Inc. © 2006 Bio-Rad Laboratories, Inc.
I Microplate sealing strips, 10
I Instruction manual
Note: The volume of supplied reagents allow for processing of the 96-well filtration
plate in a maximum of two runs (2 x 48 samples). ProteinChip serum fractionation kit
replacement buffers (catalog #K10-00008) are available to order.
Materials Needed but Not Included
I V-bottom 96-well microplate labeled samples
I V-bottom 96-well microplates labeled F1–F6
I 96-well microplate for collection of waste
I Adhesive sealing film for microplates
I 12-column partitioned buffer reservoir
I Pipet tips
I MicroMix 5 plate and tube shaker (Diagnostic Products Corp.)
I Biomek 3000 workstation integration package
(catalog #Z33-00030)
I Vacuum manifold (for manual use)
Storage
Table 1. Storage conditions for kit components.
Item Storage
ProteinChip U9 buffer –20 to –50°C
Rehydration and wash buffers 2–8°C
ProteinChip Q filtration plate 2–8°C
Rehydration Add 200 µl rehydration
buffer to each well of filtration
plate; mix 60 min at room
temperature (RT)
Remove buffer
Add 200 µl rehydration
buffer to each well
Washes
Remove buffer
Equilibration Add 200 µl U1 buffer
to each well
Washes
Remove buffer
Sample Add 50 µl sample to each well
binding
Add 50 µl sample and rinse to
each well; mix 30 min at 4°C
Fractionation Apply vacuum
and collect eluent
Add 100 µl wash buffer 1 to each well;
mix 10 min at room temperature; apply
vacuum and collect eluent
Add 100 µl wash buffer 2 to each well;
mix 10 min at room temperature;
apply vacuum and collect eluent;
repeat and pool eluents
Repeat double washes with
wash buffers 3–6, pooling
eluents for each wash buffer
Analyze fractions on
ProteinChip arrays
Bring sample
to RT
Spin
Add 20 µl sample
to each well
Add 30 µl
ProteinChip U9
buffer to each well
Mix 20 min
at 4°C
Add 50 µl U1 buffer
to each well; mix
Flow-through fraction
Fraction 1 (pH 9)
Fraction 2 (pH 8)
Fraction 3–6
Protocol Flow Chart
Sample
Preparation