Luminex 100 User Manual Version 1.7 - Manual (Page 116)

Luminex 100 User Manual Version 1.7 x MAP Technology
9 - 6 PN 89-00002-00-063 Rev. A
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.
11. Incubate the suspension for 20 ± 2 minutes in the dark at room
temperature.
12. Centrifuge the activated xMAP microspheres for 1 minute at
8,000 × g or higher. Aspirate the supernatant (See Technical note
3).