ProteinChip H50 Array (Reverse Phase) Instruction Manual

ProteinChip H50 Array (Reverse Phase) Instruction Manual
ProteinChip H50 Array
(Reverse Phase)
®
Instruction Manual
Catalog #C57-30065
For technical support,
call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD
(1-800-424-6723).
Uses
n
Protein profiling and biomarker discovery
n
Rapid protein analysis to determine purity, mass confirmation,
or both
How It Works
The ProteinChip H50 array surface binds proteins through reversephase or hydrophobic interaction chromatography and has binding
characteristics similar to that of a C6 to C12 alkyl chromatographic
resin. In reverse-phase interactions, proteins within the sample are
partitioned between the lipophilic phase of the array surface and
the sample buffer. Proteins less hydrophobic relative to the binding
buffer will not bind to the array surface, while proteins more
hydrophobic will bind to the array surface.
By increasing the organic content of the washing buffer, the
hydrophobic nature of the buffer increases. Proteins that had
previously bound to the array will repartition into the washing buffer
and be washed away if their hydrophobicity is less than that of the
washing buffer. Only the most hydrophobic proteins will be retained
with wash buffers containing a high organic solvent.
Hydrophobic interaction chromatography is characterized by
binding of proteins to a hydrophobic surface at high salt
concentrations (salt precipitation of proteins). Typically conditions
are nondenaturing, and since no organic solvent is used, biological
activity has a much higher probability of being retained. Proteins are
sequentially washed from the array surface by decreasing the salt
concentration of the wash buffers.
Packaging and Storage
Store the arrays at room temperature.
ProteinChip arrays are packaged in a 12-array cassette.
A bioprocessor reservoir is included in the package (see Figure 1).
The spare ProteinChip cassette included to separate the reservoir
from the arrays should be removed before use in the ProteinChip
cassette-compatible bioprocessor (catalog #C50-30011). It is not
necessary to remove the arrays when using the cassette-compatible
© 2006 Bio-Rad Laboratories, Inc.
bioprocessor; however, individual arrays can be removed if needed.
To do this, remove the bioprocessor reservoir before taking any arrays
out of the cassette. Be careful not to touch the spots on the array.
A pair of ProteinChip array forceps (catalog #C20-10002) helps
effectively remove the arrays from the cassette (see Figure 2).
Fig. 1. ProteinChip cassette and
reservoir.
Fig. 2. Removal of ProteinChip arrays
from cassette using array forceps.
Technical Considerations
n
Increasing the concentration of organic solvent in the
binding/washing solution will increase the selectivity of the
surface (only the most hydrophobic proteins will be retained with
higher organic solvent concentrations). Use a shorter wash time
(2 minutes or less) during the wash step after sample binding if
the washing solution contains more than 20% organic solvent
n
Increasing the salt concentration will increase hydrophobic
interactions and therefore can be included in the binding buffer.
Suggested salt concentration range is 50–1000 mM. Higher salt
concentrations are likely to adversely affect reproducibility
n
Prewashing the ProteinChip array in 50% acetonitrile or
methanol before sample binding may increase spot-to-spot
reproducibility
n
To obtain optimum performance, prewet the spot with 5 µl of
binding buffer before applying sample
n
When using a bioprocessor, make sure there are no air bubbles
in the wells. To avoid introducing bubbles, lower the pipet tip
very close to the spot surface while dispensing sample. Empty
the wells completely between washes
© 2006 Bio-Rad Laboratories, Inc.
Recommended Binding and Washing Solutions
n
ProteinChip H50 buffer (catalog #K20-00001)
(10% acetonitrile, 0.1% trifluoroacetic acid (TFA))
n
0–50% methanol or acetonitrile ± 0.1–1% TFA
n
0.1% TFA may be added to the binding solution to
increase binding
Fractionation for Serum Profiling
Fractionation of serum prior to profiling is recommended. The
fractionation procedure produces 6 fractions containing proteins
separated on the basis of their isoelectric point. Fractionation
allows segregation of highly abundant proteins into a limited
number of fractions, thereby reducing signal suppression effects
on lower-abundance proteins.
© 2006 Bio-Rad Laboratories, Inc.
Protocol 1: Serum Profiling Using the Bioprocessor
Note: These protocols are intended as a guideline; you may need to optimize the
method for your particular sample type and experimental design.
Note: This protocol is for the 8-spot array in the ProteinChip cassette-compatible
bioprocessor. For processing a single array, use the ProteinChip 8-well bioprocessor
(catalog #C50-30008).
1. Place the ProteinChip array cassette in the bioprocessor, and
prewash the arrays by adding 50 µl 50% methanol or
acetonitrile for five minutes. Repeat once.
2. Remove the prewash solution from the wells and add
150–250 µl binding solution to each well. Incubate for
5 minutes at room temperature with vigorous shaking (e.g.,
250 rpm, or on MicroMix shaker setting 20/7). Repeat once.
3. Remove the buffer from the wells. Immediately add 50–150 µl
sample to each well. Recommended concentration is
50–2,000 µg/ml total protein, in binding buffer. Incubate with
vigorous shaking for 30 minutes.
4. Remove the samples from the wells and wash each well with
150–250 µl binding buffer for 5 minutes, with agitation. Repeat
two more times.
5. Remove the binding buffer from the wells and add 150–250 µl
deionized (DI) water to each well. Remove immediately.
6. Remove the reservoir from the bioprocessor base clamp
assembly.
7. Air-dry the arrays for 5–10 minutes.
8. Add ProteinChip energy absorbing molecules (EAM) after
removing the reservoir; use the cassette hold-down frame
provided with the ProteinChip cassette-compatible
bioprocessor to keep the cassette flat during EAM addition.
9. Apply 1 µl of ProteinChip EAM in solution to each spot. Air-dry
for 5 minutes, and apply another 1 µl of EAM in solution. Allow
to air-dry.
10. Analyze the arrays using the ProteinChip SELDI system.
© 2006 Bio-Rad Laboratories, Inc.
Protocol 2: Serum Profiling On-Spot
1. Optional: Bulk-wash the ProteinChip array with 50% methanol or
acetonitrile for five minutes. Repeat once. Dry the array for an
hour after bulk wash to minimize any spot-to-spot
cross-contamination.
2. Prewet the spots with 5 µl of binding buffer for 2 minutes.
Repeat once.
3. Remove the prewetting solution and replace with 5 µl of sample.
Do not allow the spot to air-dry during sample application.
4. Incubate in a humid chamber for 30 minutes with shaking
(on MicroMix shaker setting 20/4).
5. Wash each spot with 5 µl binding buffer for 2 minutes with
shaking, and remove buffer. Repeat two more times.
6. Optional: If binding buffer contains salt, wash each spot with 5 µl
of DI water.
7. Air-dry the array for 5–10 minutes.
8. Apply 1 µl of ProteinChip EAM in solution to each spot. Air-dry
for 5 minutes, and apply another 1 µl of EAM in solution. Allow
to air-dry.
9. Analyze the array using the ProteinChip SELDI system.
© 2006 Bio-Rad Laboratories, Inc.
Ordering Information
Catalog #
Description
C57-30065
C50-30011
ProteinChip H50 Arrays, A–H format, 12
ProteinChip Cassette-Compatible Bioprocessor, includes
ProteinChip array forceps, cassette hold-down frame, 12 blank
ProteinChip arrays
ProteinChip 8-Well Bioprocessor, A–H format
ProteinChip Cassette-Compatible Bioprocessor Reservoirs, 5
ProteinChip Array Forceps, 1 pair
ProteinChip CHCA Energy Absorbing Molecules (EAM),
5 mg/vial, 20
ProteinChip SPA Energy Absorbing Molecules (EAM),
5 mg/vial, 20
ProteinChip EAM-1 Energy Absorbing Molecules (EAM),
5 mg/vial, 20
ProteinChip H50 Buffer, 200 ml
ProteinChip H50 Buffer, 1 L
C50-30008
C50-30012
C20-10002
C30-00001
C30-00002
C30-00003
K20-00001
K20-00005
© 2006 Bio-Rad Laboratories, Inc.
MicroMix is a trademark of Diagnostic Products Corporation.
The SELDI process is covered by US patents 5,719,060, 6,225,047, 6,579,719,
6,818,411, and other issued patents and pending applications in the US and other
jurisdictions.
Bio-Rad
Laboratories, Inc.
Web site www.bio-rad.com USA 800 4BIORAD
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Life Science
Group
10008230
US/EG
Rev G
06-0667
1006
Sig 1205
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