Nonmagnetic Beads and Related Reagents
Bio-Plex Pro™Magnetic COOH Beads
Bio-Plex
®
COOH Beads
Amine Coupling Kit
Instruction Manual
For technical service, call your local Bio-Rad office, or in the U.S. call 1-800-424-6723.
For research use only. Not for diagnostic procedures.
Table of Contents
Section 1 Introduction 1
Section 2 Principle 4
Section 3 Product Description 6
Section 4 Storage and Stabililty 9
Section 5 Recommended Materials 10
Section 6 Protein Preparation 12
Section 7 Protein Coupling 14
Section 7.1 Sample Protocol Using Bio-Plex Pro
Magnetic COOH Beads 14
Section 7.2 Sample Protocol Using Bio-Plex
COOH (Non magnetic) Beads 17
Section 8 Oligonucleotide Buffers 20
Section 9 Coupling of Amine-Modified Oligonucleotides 21
Section 9.1 Sample Protocol Using Bio-Plex Pro
Mag Plex COOH Beads 21
Section 9.2 Sample Protocol Using Bio-Plex
COOH Beads 24
Section 10 Validation of Coupling 26
Section 11 Bio-Plex
Section 12 Troubleshooting Guide 31
Section 13 Safety Considerations 33
Section 14 References 34
Section 15 Legal Notices 34
®
Suspension Array System Operation 28
Section 1
Introduction
The Bio-Plex Pro Magnetic COOH Beads and Bio-Plex COOH Beads are
suited for coupling to proteins or nucleic acids. Bio-Plex Pro Magnetic Beads
are identical to MagPlex
identical to Microplex Microspheres, both developed by Luminex corporation.
This instruction manual describes the coupling procedures.
The Bio-Plex Amine Coupling Kit (catalog #171-406001) contains all of the
buffers necessary to covalently couple 6–150 kD proteins to 6.5 µm Bio-Plex
Pro Magnetic COOH beads (magnetic, carboxylated) or 5.5 µm Bio-Plex
COOH beads (nonmagnetic, carboxylated) in under three hours. Coupling is
achieved via carbodiimide reactions involving the primary amino groups on the
protein and the carboxyl functional groups on the bead surface. The covalent
attachment is permanent, leaving no unbound protein after cleanup, even after
months of storage. The contents of the kit are sufficient for 30 coupling
reactions. Each coupling reaction requires 1.25 x 10
scale). For larger scale coupling reactions, the volume of the buffers used in
each step can be proportionally increased to a maximum 10x scale. The
protein-coupled beads can then be used in multiplex protein-protein binding
studies or in the development of multiplex assays that can be analyzed with the
Bio-Plex suspension array system. The bead yield per coupling reaction is
approximately 80%, enough protein-coupled beads for four 96-well microtiter
plates using 2,500 beads per well.
®
Microspheres, and Bio-Plex COOH Beads are
6
carboxylated beads (1x
For nucleic acid oligonucleotide coupling, refer to this manual for ordering
information and buffer recipes. The amine coupling kit contains buffers used for
coupling proteins only.
The coupling accessories for multiplex assay development complement the
Bio-Plex assay products and associated kits. For a current listing of Bio-Plex
products,visit us on the Web at www.bio-rad.com/bio-plex.
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2
3
Section 2
Principle
Amine Coupling
The coupling procedure involves a two-step carbodiimide reaction. The carboxyl
groups on the surface of the polystyrene beads must first be activated with a
carbodiimide derivative prior to coupling the protein. EDAC (1-ethyl-3-[3dimethylaminopropyl] carbodiimide hydrochloride) reacts with carboxyl groups
on the surface of the beads to form an active O-acylisourea intermediate. This
intermediate forms a more stable ester using S-NHS (Nhydroxysulfosuccinimide). The ester reacts with the primary amines (NH
of proteins or amine-modified oligonucleotides to form a covalent bond (amide
linkages).
A number of buffers can be used successfully in this coupling reaction. As no
buffer is ideal for every ligand, the protocols provided in this manual do not
contain recommendations for specific buffers. Generally the pH at which a
coupling reaction occurs should be compatible to the solubility of the ligand of
interest. PBS and MES buffers are two popular choices mentioned in this
manual. PBS buffer is provided in the kit. MES buffer can be prepared according
to the formulation provided in Section 8 for the coupling of oligonucleotides.
groups)
2
Protein Preparation
This coupling procedure can be used to covalently couple water soluble
proteins ranging in size from 6–150 kD via carboxyl groups on the surface of
the beads. The protein sample must be free of sodium azide, BSA, glycine, Tris,
or amine-containing additives and must be suspended in PBS, pH 7.4. Optimal
protein coupling conditions must be established. First determine how much
protein will be required for the coupling reaction to promote optimal binding
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between the bound protein and its complementary ligand in the protein assay.
The table below provides examples of optimal amounts per coupling reaction
for four different proteins. Note that using the highest level of protein for the
coupling reaction will not necessarily yield an optimal assay. The ultimate test is
the functional assay for each coupled protein.
Example: Optimal amount of protein for one coupling reaction
Protein
Insulin
Human IL-10
Erk
Mouse IgG
Protein Coupling Validation
Once the coupling reaction has been completed, the protein-coupled beads are
enumerated and the efficiency of the protein coupling reaction is validated. In this
procedure, the protein-coupled beads are reacted with a phycoerythrin (PE)labeled antibody that binds to the coupled protein, which is then analyzed using
the Bio-Plex suspension array system. This procedure may be performed by
reacting the beads with a PE-labeled antibody; alternatively, a reaction using a
biotinylated antibody followed by streptavidin-PE may be used. The intensity of
the fluorescent signal of this reaction is directly proportional to the amount of
protein on the surface of the beads. The protein coupling validation procedure
provides a rapid assessment of the relative amount of protein coupled to the
beads; however, this procedure does not verify the functionality of the protein.
MW (kD)
6
18.6
44
150
Mass (μg)
5
2
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9
5
Section 3
Product Description
Bio-Plex Pro Magnetic COOH Beads
For magnetic or vacuum separation methods
All regions are at a concentration of 1.25 x 10
MC10026-01 Bio-Plex Pro Magnetic COOH Beads, Region 26, 1 ml
MC10027-01 Bio-Plex Pro Magnetic COOH Beads, Region 27, 1 ml
MC10028-01 Bio-Plex Pro Magnetic COOH Beads, Region 28, 1 ml
MC10029-01 Bio-Plex Pro Magnetic COOH Beads, Region 29, 1 ml
MC10034-01 Bio-Plex Pro Magnetic COOH Beads, Region 34, 1 ml
MC10035-01 Bio-Plex Pro Magnetic COOH Beads, Region 35, 1 ml
MC10036-01 Bio-Plex Pro Magnetic COOH Beads, Region 36, 1 ml
MC10037-01 Bio-Plex Pro Magnetic COOH Beads, Region 37, 1 ml
MC10043-01 Bio-Plex Pro Magnetic COOH Beads, Region 43, 1 ml
MC10044-01 Bio-Plex Pro Magnetic COOH Beads, Region 44, 1 ml
MC10045-01 Bio-Plex Pro Magnetic COOH Beads, Region 45, 1 ml
MC10046-01 Bio-Plex Pro Magnetic COOH Beads, Region 46, 1 ml
MC10049-01 Bio-Plex Pro Magnetic COOH Beads, Region 49, 1 ml
MC10052-01 Bio-Plex Pro Magnetic COOH Beads, Region 52, 1 ml
MC10053-01 Bio-Plex Pro Magnetic COOH Beads, Region 53, 1 ml
MC10054-01 Bio-Plex Pro Magnetic COOH Beads, Region 54, 1 ml
MC10055-01 Bio-Plex Pro Magnetic COOH Beads, Region 55, 1 ml
MC10062-01 Bio-Plex Pro Magnetic COOH Beads, Region 62,1 ml
MC10063-01 Bio-Plex Pro Magnetic COOH Beads, Region 63, 1 ml
MC10064-01 Bio-Plex Pro Magnetic COOH Beads, Region 64, 1 ml
MC10065-01 Bio-Plex Pro Magnetic COOH Beads, Region 65, 1 ml
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beads/mL
Additional bead regions and larger volumes are available with longer delivery times.
Call your local Bio-Rad Technical Service center for more information.
NOTE: The following bead regions are not compatible with the Bio-Plex
100/200 or Luminex 100/200 systems: 1 –5, 10, 11, 16, 17, 23, 24, 31,
32, 40, 41, 50, 60, 71, 92, and all regions over 100.
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Bio-Plex COOH Beads
For vacuum separation methods only.
All regions are at a concentration of 1.25 x 10
171-506011 Bio-Plex COOH Beads, Region 11, 1 ml
171-506016 Bio-Plex COOH Beads, Region 16, 1 ml
171-506018 Bio-Plex COOH Beads, Region 28, 1 ml
171-506020 Bio-Plex COOH Beads, Region 20, 1 ml
171-506024 Bio-Plex COOH Beads, Region 24, 1 ml
171-506025 Bio-Plex COOH Beads, Region 25, 1 ml
171-506026 Bio-Plex COOH Beads, Region 26, 1 ml
171-506027 Bio-Plex COOH Beads, Region 27, 1 ml
171-506028 Bio-Plex COOH Beads, Region 28, 1 ml
171-506030 Bio-Plex COOH Beads, Region 30, 1 ml
171-506031 Bio-Plex COOH Beads, Region 31, 1 ml
171-506033 Bio-Plex COOH Beads, Region 33, 1 ml
171-506035 Bio-Plex COOH Beads, Region 35, 1 ml
171-506038 Bio-Plex COOH Beads, Region 38, 1 ml
171-506042 Bio-Plex COOH Beads, Region 42, 1 ml
171-506043 Bio-Plex COOH Beads, Region 43, 1 ml
171-506044 Bio-Plex COOH Beads, Region 44, 1 ml
171-506045 Bio-Plex COOH Beads, Region 45, 1 ml
171-506046 Bio-Plex COOH Beads, Region 46, 1 ml
171-506050 Bio-Plex COOH Beads, Region 50, 1 ml
171-506051 Bio-Plex COOH Beads, Region 51, 1 ml
171-506052 Bio-Plex COOH Beads, Region 53, 1 ml
171-506053 Bio-Plex COOH Beads, Region 53, 1 ml
171-506056 Bio-Plex COOH Beads, Region 56, 1 ml
171-506066 Bio-Plex COOH Beads, Region 66, 1 ml
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beads/mL
Additional bead regions and larger volumes are available with longer delivery times.
Call your local Bio-Rad Technical Service center for more information.
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Required Reagents for Protein Coupling
Bio-Plex Amine Coupling Kit (catalog #171-406001)
Bead wash buffer
Bead activation buffer
PBS, pH 7.4
Blocking buffer
Storage buffer
Staining buffer
Coupling reaction tubes
Required Reagents for Oligonucleotide Coupling
See Section 8 for buffer recipes
Other Required Reagents
EDAC coupling reagent, 5g (catalog #153-0990)
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Section 4
Storage and Stability
Protect the photosensitive beads from light by storing them in the
resealable foil bags provided. All kit components should be stored at 4°C.
Do not freeze.
All components are guaranteed for 6 months from the date of purchase
when stored as specified in this manual.
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Section 5
Recommended Materials
For optimal results we strongly recommend the use of the following:
Bio-Plex Suspension Array System
See bulletin 5405 or go to www.bio-rad.com/bio-plex for ordering
information
Bio-Plex Suspension Array System Accessories
Bio-Plex validation kit, Bio-Rad catalog #171-203000
Bio-Plex calibration kit, Bio-Rad catalog #171-203060
Bio-Plex Pro wash station (for magnetic washing only), Bio-Rad catalog
#300-34376
Bio-Plex Pro II wash station (for magnetic or vacuum washing), Bio-Rad
catalog #300-34377
Bio-Rad Aurum vacuum manifold (alternative for vacuum washing),
Bio-Rad catalog #732-6470
Magnetic separator for 1.5 mL tubes
Dynal MPC-S magnetic bead separator, Dynal Biotech catalog #120-20D
Dynal MPC-S magnetic bead separator for 1.5 mL tubes, Dynal Biotech
(cat#120-20D)
Dynal MPC-S magnetic bead separator for 15 mL tubes, Dynal Biotech
(cat#120-01D)
Magnetic Separators for Round-Bottom Plates
96-well plate magnet, PerkinElmer catalog #5083175
LifeSep 96F, Dexter Magnetic Technologies catalog #2501008
Vortexer
VWR vortexer mini, VWR catalog #58816-121
Centrifuge
Microcentrifuge model 5415C, VWR catalog #20901-051
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