Luminex 100 IS Developer Workbench Guide Version 2.3 - Manual (Page 60)

Luminex 100 IS Developer Workbench Guide Version 2.3
x
MAP Technology
54 PN 89-00002-00-084 Rev. B
Coupling,
Blocking, and
Storage
1. Add the protein preparation to the resuspended microspheres.
2. Bring total volume to 500 µL with Coupling Buffer. Vortex.
3. Rotate the mixture for 2 hours in the dark at room temperature.
4. Centrifuge the microspheres 1 to 2 minutes at
≥ 8,000 × g.
Aspirate the supernatant.
5. If you plan to use the coupled microspheres on the day of
coupling, resuspend the microspheres in 500 µL of Blocking/
Storage Buffer. Vortex and sonicate for approximately 20
seconds. Rotate for 30 minutes in the dark at room temperature
to block the coupled microspheres. Centrifuge the microspheres
1 to 2 minutes at
≥ 8,000 × g. Aspirate the supernatant.
6. If you plan to use the coupled microspheres at a later day,
resuspend the microspheres in 500 µL of Wash Buffer. Vortex
and sonicate for approximately 20 seconds.
7. Centrifuge the microspheres 1 minute at
≥ 8,000 × g for 1 to 2
minutes.
8. Aspirate the supernatant. Resuspend the microspheres in 1 mL of
Wash Buffer. Vortex and sonicate for about 20 seconds.
9. Centrifuge the microspheres 1 minute at
≥ 8,000 × g for 1 to 2
minutes.
10. Repeat steps 8 and 9 for a total of two washes with Wash Buffer.
11. Resuspend the coupled and washed microspheres in 500 µL of
Blocking/Storage Buffer.
12. Count the microsphere suspension by hemacytometer.
Calculation: Total microspheres = Count (1 corner of 4 x 4
section) x (1 x 10
4
) x (dilution factor) x (resuspension volume in
mL).
13. Store the preparation at 2
o
C - 8
o
C. Protect it from light.
See note 2.
Technical Notes
1. For best results, use microcentrifuge tubes from USA Scientific,
Inc., catalog number 1415-2500. To order, call 1-800-LAB-TIPS.
2. Coupling can be performed in 100 mM MES, pH 6.0 with
similar results. For some proteins, better solubility and better
coupling may be achieved at a higher coupling pH or in a