Luminex 100 User Manual Version 1.7 User Manual

Page 117

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MAP Technology

Protocols

PN 89-00002-00-063 Rev. A

9 - 7

13. Add 500 µL of COUPLING BUFFER to the activated xMAP

microspheres and vortex. Centrifuge the activated xMAP
microspheres for 1 minute at 8,000

× g or higher. 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 xMAP microspheres; vortex.

2. Rotate the mixture for 2 hours in the dark at room temperature.

3. Centrifuge the protein-coupled xMAP microspheres 1 minute at

8,000

× g or higher. Aspirate the supernatant.

4. Add 1 mL of WASH BUFFER to the coupled xMAP

microspheres and vortex. Centrifuge the protein-coupled
microspheres 1 minute at 8,000

× g or higher. Aspirate the

supernatant.

5. Repeat step 4.

6. Suspend the xMAP microsphere pellet in a desired volume of

BLOCKING/STORAGE BUFFER.

7. Enumerate the xMAP 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 xMAP microspheres may form a

sheet-like layer on the side of the microfuge tube. This 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.

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