Luminex 100 IS Developer Workbench Guide Version 2.3 - Manual (Page 59)

PN 89-00002-00-084 Rev. B 53
x
MAP Technology Protein Coupling Protocol
Preparation
1. Allow all reagents to warm to room temperature.
2. Using an analytical balance, weigh approximately 10 mg of
Sulfo-NHS into a tube. Repeat for EDC.
Procedure
xMAP Microsphere
Activation
1. Vortex and sonicate the stock microspheres for 20 seconds.
2. Transfer 5.0
× 10
6
of the stock microspheres to a USA Scientific
microfuge tube.
3. Centrifuge the stock microspheres at
≥ 8,000 × g for 1 to 2
minutes.
4. Aspirate the supernatant and resuspend the pelleted microspheres
in 100 µL dH
2
O by sonication for approximately 20 seconds.
5. Centrifuge the stock microspheres at
≥ 8,000 × g for 1 to 2
minutes.
6. Aspirate the supernatant. Resuspend the washed microspheres in
80 µL of Activation Buffer. Vortex and sonicate for
approximately 20 seconds.
7. Add 10 µL of 50 mg/mL Sulfo-NHS (diluted in Activation
Buffer or dH
2
O) to the microspheres and mix gently by vortex.
8. Add 10 µL of 50 mg/ML EDC (diluted in Activation Buffer or
dH
2
O) to the microspheres and mix gently by vortex.
9. Incubate for 20 minutes at room temperature with gentle mixing
by vortex at 10 minute intervals.
10. Centrifuge the activated microspheres at
≥ 8,000 × g for 1 to 2
minutes.
11. Aspirate the supernatant and resuspend the microspheres in 250
µL of Coupling Buffer by vortex and sonication for
approximately 20 seconds (see Technical note 5).
12. Centrifuge the microspheres for 1 to 2 minutes at
≥ 8,000 × g.
13. Repeat steps 11 and 12 for a total of two washes with Coupling
Buffer.
14. Resuspend the activated and washed microspheres in 100 µL of
Coupling Buffer. Vortex and sonicate for approximately 20
seconds.