Bio-Rad Nonmagnetic Beads and Related Reagents - Manual (Page 28)

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Section 10
Validation of Coupling
This validation method is based on the detection of the coupled protein
with labeled antibodies. This procedure describes two validation methods,
using either a PE-conjugated antibody or a biotinylated antibody followed
by streptavidin-PE.
Note: If an antibody is coupled to the beads, ensure that the antibody
used in this procedure is specific for the host species of your coupled
antibody. For example, if you have coupled a mouse anti-human antibody,
your PE-labeled antibody should be directed against the mouse antibody
(i.e. goat anti-mouse or rabbit anti-mouse).
1. Label two microcentrifuge tubes for each bead coupled, one as the
negative control and one as the test.
2. Vortex the coupled beads at medium speed for 15 sec. Add
approximately 5,000 coupled beads to each of the two tubes.
3. a) When using a PE-conjugated antibody, dilute the PE-labeled
antibody to 1 µg/ml with staining buffer. Add 50 µl of the 1 µg/ml
diluted PE-labeled antibody to the tube labeled “test”. Do not
add antibody to the negative control tube. Cover the tubes with
aluminum foil and agitate the beads with a shaker or a rotator at
room temperature for 30 min.
Or
b) When using a biotinylated antibody followed by streptavidin-PE,
dilute the biotinylated antibody to 2 µg/ml with staining buffer.
Add 50 µl of staining buffer to the negative control tube. Add
50 µl of the diluted biotinylated antibody to the tube labeled
“test”. Do not add antibody to the negative control tube. Cover
the tubes with aluminum foil and agitate the beads with a rotator
at room temperature for 30 min.