Luminex xMAP Antibody Coupling Kit - Manual (Page 16)

Page 12 of 20
20. Calculate the volume of Activation Buffer needed for the reaction.
• If coupling more than 5x10
6
microspheres, subtract the volume of antibody
calculated in Step 19 from 1000 μL.
• If coupling 5x10
6
microspheres or less, subtract the volume of antibody
calculated in Step 19 from 500 μL.
Example Calculation:
If 5 μL of the stock antibody is needed (as calculated in the example in Step 19) for
the coupling of 5 million microspheres:
Volume of Activation Buffer needed = (1000μL or 500μL) - (Volume of Ab needed)
Volume of Activation Buffer needed = 500μL - 5μL
Volume of Activation Buffer needed = 495μL
21. Add the appropriate volume of Activation Buffer, calculated in Step 20, to the
reaction tube.
22. Add the appropriate volume of antibody, calculated in Step 19, to the reaction tube.
23. Vortex the reaction tube for a minimum of 10 seconds.
24. Protect microspheres from light and rotate on rotator for 2 hours. (Rotation speed
should be ~15-30 rpm)
25. Wash the microspheres.
A. Place the reaction tube with microspheres into the magnetic separator for 1-2
minutes.
B. With the tube still positioned in the magnetic separator, remove the supernatant
with a transfer pipette.
C. Add 500 μL of Wash Buffer into the reaction tube.
D. Vortex the reaction tube for 10 seconds and then sonicate for 10 seconds to
disperse the microspheres.
26. Repeat Step 25 twice, for a total of three washes.
27. 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.
28. Add 1 mL of Wash Buffer into the reaction tube.
NOTE: The Wash Buffer is used as a storage buffer after completing the coupling
reaction.
29. Vortex the reaction tube for 10 seconds and then sonicate for 10 seconds to
disperse the microspheres.
30. Protect from light and store at 2-8 ºC until needed.