Luminex 100 IS Version 2.2 - Manual (Page 85)

PN 89-00002-00-032 Rev. A 79
x MAP Technology
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
Microsphere activation.
1. Centrifuge the x MAP microsphere stock for 1 minute at 10,000
× g.
2. Disperse the microsphere pellet with sonication, and vortex the
container for 20 seconds.
3. Dispense 2.5 × 10
6
microspheres from homogeneous x MAP
microsphere stock into a 1.5 mL microcentrifuge tube.
4. Centrifuge the reaction tube for 1 minute at 10,000 × g. Aspirate
the supernatant.
5. Wash twice with 80 µL ACTIVATION BUFFER.
6. Resuspend the microspheres in 80 µL of ACTIVATION
BUFFER and sonicate the tube until a homogeneous distribution
of 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 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 microsphere suspension.
Vortex gently.
11. Incubate the suspension for 20 ± 2 minutes in the dark at room
temperature.
12. Centrifuge the activated microspheres for 1 minute at 10,000 × g.
Aspirate the supernatant (See Technical note 3).