Luminex 100 IS Version 2.1 - Manual (Page 76)

Luminex 100 IS Developer Guide to xMAP Technology Version 2.1 x MAP Technology
70 PN 89-00002-00-062 Rev. A
13. Add 500 µL of COUPLING BUFFER to the activated
microspheres and vortex. Centrifuge the activated microspheres
for 1 minute at
≥ 8,000 × g. Aspirate the supernatant.
14. Repeat the previous step and go immediately to the protein
coupling steps.
Coupling,
blocking, and
storage
1. To initiate the coupling reaction, add 500 µL of the protein
preparation (protein stock in COUPLING BUFFER) to the
activated microspheres; vortex.
2. Rotate the mixture for 2 hours in the dark at room temperature.
3. Centrifuge the protein-coupled microspheres 1 minute at
≥ 8,000 × g. Aspirate the supernatant.
4. Add 1 mL of WASH BUFFER to the coupled microspheres and
vortex. Centrifuge the protein-coupled microspheres 1 minute at
≥ 8,000 × g. Aspirate the supernatant.
5. Repeat step 4.
6. Suspend the microsphere pellet in a desired volume of
BLOCKING/STORAGE BUFFER.
7. Enumerate the microsphere preparation.
8. Store the preparation at 2
o
C - 8
o
C. Protect it from light.
Technical Notes
1. Minimize the exposure of EDC and Sulfo-NHS to air; secure
closures on stock and aliquot containers. Use aliquots
immediately and discard containers after use.
2. The optimal coupling concentration for a given protein is
determined by coupling at various concentrations within the
recommended range. For best results, the protein stock should
not contain foreign protein, azide, glycine, Tris, or any primary
amines. If any of these agents exist in the protein preparation,
remove them through dialysis or chromatography.
3. During activation, centrifuged microspheres may form a sheet-
like layer on the side of the microfuge tube. This formation is
normal.
4. For best results, we recommend using microcentrifuge tubes
from USA Scientific, Inc., catalog number 1415-2500. To order,
call 1-800-LAB-TIPS.