ProteinChip®Energy
Absorbing Molecules
(EAM)
Instruction Manual
Catalog #C30-00001, #C30-00002,
#C30-00003, #C30-00004
For technical support,
call your local Bio-Rad office, or
in the US, call 1-800-4BIORAD
(1-800-424-6723).
Introduction
ProteinChip energy absorbing molecules (EAM) are an
essential component of successful ProteinChip SELDI
system experiments. EAM is a generic name for
molecules that assist in desorption and ionization of the
analyte. Known as “matrix” in traditional MALDI mass
spectrometry, EAM are applied in organic solvent,
solubilizing many proteins on the array surface. As the
EAM dry, the proteins cocrystallize with the EAM. These
crystals absorb the laser energy and generate the ionized
proteins detected by the ProteinChip SELDI system.
Product Description
Bio-Rad offers three EAM for the detection of proteins
and peptides. The general guidelines for choosing EAM
are based on the molecular weight (MW) and chemical
nature of the analyte, but there are no absolute rules.
ProteinChip CHCA EAM, 5 mg/vial
ProteinChip alpha-cyano-4-hydroxy cinnamic acid
(CHCA) EAM (MW 189.2) are especially good for small
molecules, 1–30 kD.
ProteinChip SPA EAM, 5 mg/vial
ProteinChip sinapinic acid (SPA) EAM (MW 224.2)
are recommended for all larger proteins, but also
work reasonably well for peptides. In general,
© 2006 Bio-Rad Laboratories, Inc.
ProteinChip SPA EAM give better resolution and fewer
multiply charged ions than ProteinChip EAM-1 EAM.
For protein profiling using only one kind of EAM,
ProteinChip SPA EAM are the molecules of choice.
ProteinChip EAM-1 EAM, 5 mg/vial
ProteinChip EAM-1 EAM are proprietary molecules
(MW 231.21) and work well for proteins in the 10–50 kD
mass range. ProteinChip EAM-1 EAM prove useful in
allowing desorption and ionization of proteins that are
difficult to detect, such as glycosylated proteins. It
should be noted that ProteinChip EAM-1 EAM tend to
generate multiply charged species, and peaks tend to
be broader due to more adduct formation.
Preparing ProteinChip EAM in Solution
ProteinChip EAM in solution can be prepared in a
number of different ways that may optimize the
detection of the proteins in your sample. Table 1 to the
right lists the steps for preparing EAM in solution using
commonly used solvent systems. For a comprehensive
list of solvent solution options, please see the
ProteinChip SELDI System Applications Guide,
Volume 1.
© 2006 Bio-Rad Laboratories, Inc.
Table 1. Preparation of ProteinChip EAM in solution using
commonly used solvent systems.
EAM Solvent System Preparation
ProteinChip CHCA 50% acetonitrile
49.75% HPLC
grade H
O
2
0.25% trifluoroacetic
acid (TFA)
Preparation
1. Add 200 µl solvent to vial
(there should be undissolved
CHCA in the bottom of the
tube)
2. Mix
3. Centrifuge
4. Draw off supernatant and
dilute with equal part solvent
ProteinChip SPA 50% acetonitrile
49.50% HPLC
O
grade H
2
1. Add 400 µl solvent to vial
2. Mix
3. Centrifuge
0.50% TFA
ProteinChip EAM-1 50% acetonitrile
49.50% HPLC
O
grade H
2
0.50% TFA
1. Add 200 µl solvent to vial
(there should be undissolved
EAM-1 in the bottom of the
tube)
2. Mix
3. Centrifuge
4. Draw off supernatant and
dilute with 2x volume of
solvent
© 2006 Bio-Rad Laboratories, Inc.
Solvent systems and ProteinChip EAM in solution are
best prepared fresh every day. Acetonitrile volatilizes
rapidly, and EAM in solution break down significantly
within 24 hours at room temperature. Freezing EAM after
dissolving them in solvent may preserve them for about a
week; however, it is recommended to prepare fresh
solvent and ProteinChip EAM in solution each day.
Preparing Saturated Solutions
1. Bio-Rad supplies ProteinChip EAM as 5 mg of dried
powder in a tube. Tap the tube to ensure that contents
fall to the bottom of the tube before reconstitution. Add
50–200 µl of the appropriate solvent depending on your
needs. Vortex for approximately 5 minutes, then let sit at
room temperature for approximately 5 minutes. There
should be undissolved EAM remaining in the tube.
2. Microcentrifuge for 10 minutes at 10,000 rpm at
room temperature to pellet any particulates. The
ProteinChip EAM are now ready to use. Keep at
room temperature. The solubility drops significantly
when kept on ice.
Note: Occasionally CHCA particulate will float on top of the solution. To
minimize deposition of solid CHCA onto the spot, take the supernatant
off the top of the CHCA and transfer it to another tube.
© 2006 Bio-Rad Laboratories, Inc.
Applying EAM to Arrays
The method of ProteinChip EAM application to the
array surface requires practice as it can significantly
influence the quality of the data. A minute volume
(0.3–1.0 µl) of ProteinChip EAM in organic solvent must
be applied to each sample prior to analysis. It is
recommended that ProteinChip EAM be applied to the
spots after they have dried completely for the greatest
spot-to-spot consistency.
If variable signal is seen across the spot when reading
the arrays, it may be useful to observe the
ProteinChip EAM in good light, or under a dissecting
microscope. The EAM should be distributed fairly
uniformly across the spot surface.
Note:
1. When pipetting ProteinChip EAM onto samples, be careful to avoid
the undissolved EAM at the bottom of the tube.
2. Always use new, packaged pipet tips; never use tips that have
been autoclaved or reused.
Storage
ProteinChip EAM are light-sensitive and should be
stored away from the light.
© 2006 Bio-Rad Laboratories, Inc.
Ordering Information
Catalog # Description
C30-00001
C30-00002
C30-00003
C30-00004
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.
ProteinChip CHCA Energy Absorbing Molecules
, 5 mg/vial, 20
(EAM)
ProteinChip SPA Energy Absorbing Molecules
(EAM)
, 5 mg/vial, 20
ProteinChip EAM-1 Energy Absorbing Molecules
(EAM)
, 5 mg/vial, 20
ProteinChip Energy Absorbing Molecules (EAM) Kit,
includes 6 vials each CHCA, EAM-1, and SPA EAM
© 2006 Bio-Rad Laboratories, Inc.
Life Science
Group
06-0680 1006 Sig 1205
10008246 US/EG Rev E
Bio-Rad
Laboratories, Inc.
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