Bio-Rad ProteinChip Serum Fractionation Kits User Manual
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he SELDI process is covered by US patents 5,719,060, 6,225,047, 6,579,719, and
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jurisdictions.
ProteinChip®Serum
Fractionation Kit
Instruction Manual
Catalog #K10-00007
For technical support,
call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD
(1-800-424-6723).
Introduction
The ProteinChip serum fractionation kit is designed to facilitate the
analysis of crude serum samples by fractionating proteins based on
their biophysical properties.
The kit allows high-throughput fractionation by using anion exchange
media in a 96-well microplate format. The anion exchange support is
supplied in a 96-well ProteinChip Q filtration plate and requires
rehydration before use. The samples are added to the plate and then
eluted in a stepwise manner by altering the pH of the wash buffer
until six fractions are collected. The fractions can then be analyzed
on a ProteinChip array using a profiling protocol. Each of the six
fractions is collected twice, and the two collections are pooled. This
helps to ensure that the pH changes appropriately, and also results in
greater reproducibility in the fractionation.
The fractions can be analyzed in your particular profiling application.
If you are using multiple array types or conditions, we recommend
that the same fraction is profiled at one time to avoid multiple
freeze-thaw cycles.
Materials
Materials Included
n
ProteinChip U9 buffer (9 M urea, 2% CHAPS, 50 mM Tris-HCl,
pH 9), 20 ml
n
Rehydration buffer (50 mM Tris-HCl, pH 9), 250 ml
n
Wash buffer 1 (50 mM Tris-HCl, 0.1% OGP, pH 9), 20 ml
n
Wash buffer 2 (50 mM HEPES, 0.1% OGP, pH 7), 30 ml
n
Wash buffer 3 (100 mM Na acetate, 0.1% OGP, pH 5), 30 ml
n
Wash buffer 4 (100 mM Na acetate, 0.1% OGP, pH 4), 30 ml
n
Wash buffer 5 (50 mM Na citrate, 0.1% OGP, pH 3), 30 ml
n
Wash buffer 6 (33.3% isopropanol, 16.7% acetonitrile,
0.1% trifluoroacetic acid), 30 ml
n
ProteinChip Q filtration plate, filled with dehydrated anion
exchange Q ceramic HyperD F sorbent, 1
© 2006 Bio-Rad Laboratories, Inc.© 2006 Bio-Rad Laboratories, Inc.
n
Microplate sealing strips, 10
n
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
eplacement buffers (catalog #K10-00008) are available to order.
r
Protocol Flow Chart
ehydrationAdd 200 µl rehydration
R
buffer to each well of filtration
plate; mix 60 min at room
temperature (RT)
Materials Needed but Not Included
n
V-bottom 96-well microplate labeled samples
n
V-bottom 96-well microplates labeled F1–F6
n
96-well microplate for collection of waste
n
Adhesive sealing film for microplates
n
12-column partitioned buffer reservoir
n
Pipet tips
n
MicroMix 5 plate and tube shaker (Diagnostic Products Corp.)
n
Biomek 3000 workstation integration package
(catalog #Z33-00030)
n
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
Remove buffer
Add 200 µl rehydration
buffer to each well
Remove buffer
Equilibration Add 200 µl U1 buffer
to each well
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
Washes
Washes
Bring sample
to RT
Spin
Add 20 µl sample
to each well
Sample
Preparation
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
Analyze fractions on
ProteinChip arrays
© 2006 Bio-Rad Laboratories, Inc.© 2006 Bio-Rad Laboratories, Inc.
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