Luminex 200 User Manual with LDS 1.7 Software - Manual (Page 123)

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MAP Technology Protocols
PN 89-00002-00-150 Rev. A 9 - 5
• Sulfo-NHS:
N-Hydroxysulfosuccinimide sodium salt, Pierce Chemicals
• EDC:
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride,
Pierce Chemicals
• Protein for coupling (See Technical note 2)
Preparation
1. Allow all reagents to warm to room temperature.
2. Dilute the protein stock with COUPLING BUFFER to a
concentration of 25 - 250 µg/ml and a volume of at least 500 µl
(See Technical note 2).
3. Using an analytical balance, weigh approximately 10 mg of
Sulfo-NHS into a tube. Repeat for EDC (See Technical note 1).
Procedure
xMAP Microsphere Activation.
1. Centrifuge the xMAP microsphere stock for 1 minute at 8,000
×
g or higher.
2. Disperse the xMAP microsphere pellet with sonication, and
vortex the container for 20 seconds.
3. Dispense 2.5
× 10
6
microspheres from homogeneous xMAP
microsphere stock into a 1.5 ml microcentrifuge tube.
4. Centrifuge the reaction tube for 1 minute at 8,000
× g or higher.
Aspirate the supernatant.
5. Wash twice with 80 µl ACTIVATION BUFFER.
6. Resuspend the xMAP microspheres in 80 µL of ACTIVATION
BUFFER and sonicate the tube until a homogeneous distribution
of xMAP microspheres is observed.
7. Immediately before use, make a 50 mg/ml Sulfo-NHS solution
by adding ACTIVATION BUFFER to the Sulfo-NHS aliquot.
Mix.
8. Add 10 µl of the Sulfo-NHS solution to the xMAP microsphere
suspension and vortex gently. Go immediately to the next step.
9. Make a 50 mg/ml EDC solution by adding ACTIVATION
BUFFER to an aliquot of EDC. Mix.
10. Add 10 µl of the EDC solution to the xMAP microsphere
suspension. Vortex gently.