Luminex 200 User Manual with LDS 1.7 Software - Manual (Page 124)

Luminex 200 User Manual for LDS Version 1.7
x
MAP Technology
9 - 6 PN 89-00002-00-150 Rev. A
11. Incubate the suspension for 20 ± 2 minutes in the dark at room
temperature.
12. Centrifuge the activated xMAP microspheres for 1 minute at
8,000
× g or higher. Aspirate the supernatant (See Technical note
3).
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