Luminex xMAP Antibody Coupling Kit - Manual (Page 14)

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7. Place the reaction tube with microspheres into the magnetic separator for 1-2
minutes and, with the tube still positioned in the magnetic separator, remove the
supernatant with a transfer pipette.
8. Add Activation Buffer into the reaction tube.
• If coupling more than 5x10
6
microspheres, add 400 μL of Activation Buffer into
the reaction tube.
• If coupling 5x10
6
microspheres or less, add 480 μL of Activation Buffer into the
reaction tube.
9. Vortex the reaction tube for 10 seconds and then sonicate for 10 seconds to
disperse the microspheres.
10. Vortex the provided Sulfo-NHS tube for a minimum of 10 seconds.
11. Add Sulfo-NHS solution to the reaction tube.
• If coupling more than 5x10
6
microspheres, add 50 μL of the Sulfo-NHS solution
into the reaction tube.
• If coupling 5x10
6
microspheres or less, add 10 μL of the Sulfo-NHS solution into
the reaction tube.
12. Add 250 μL of Activation Buffer into the 10 mg vial of EDC. Invert the EDC vial and
then vortex the vial for 10-12 seconds to dissolve the EDC.
IMPORTANT: EDC will begin to degrade once exposed to moisture in the atmo-
sphere and the Activation Buffer. Once opened the EDC solution must be pre-
pared and used quickly. If performing multiple reactions, be sure to prepare all of
them prior to this step so that the EDC solution can be quickly added to all of the
reaction tubes immediately after dissolution. The EDC solution must be made
fresh for each coupling event and the excess should be discarded.
13. Add EDC Solution to reaction tube.
• If coupling more than 5x10
6
microspheres, add 50 μL of the EDC solution into
the reaction tube.
• If coupling 5x10
6
microspheres or less, add 10 μL of the EDC solution into the
reaction tube.
14. Vortex the reaction tube for a minimum of 10 seconds.
15. Protect microspheres from light and rotate on rotator for 20 minutes.
(Rotation speed should be ~15-30 rpm)