Bio-Rad Nonmagnetic Beads and Related Reagents - Manual (Page 7)

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between the bound protein and its complementary ligand in the protein assay.
The table below provides examples of optimal amounts per coupling reaction
for four different proteins. Note that using the highest level of protein for the
coupling reaction will not necessarily yield an optimal assay. The ultimate test is
the functional assay for each coupled protein.
Example: Optimal amount of protein for one coupling reaction
Protein Coupling Validation
Once the coupling reaction has been completed, the protein-coupled beads are
enumerated and the efficiency of the protein coupling reaction is validated. In this
procedure, the protein-coupled beads are reacted with a phycoerythrin (PE)-
labeled antibody that binds to the coupled protein, which is then analyzed using
the Bio-Plex suspension array system. This procedure may be performed by
reacting the beads with a PE-labeled antibody; alternatively, a reaction using a
biotinylated antibody followed by streptavidin-PE may be used. The intensity of
the fluorescent signal of this reaction is directly proportional to the amount of
protein on the surface of the beads. The protein coupling validation procedure
provides a rapid assessment of the relative amount of protein coupled to the
beads; however, this procedure does not verify the functionality of the protein.
Protein
Insulin
Human IL-10
Erk
Mouse IgG
MW (kD)
6
18.6
44
150
Mass (
μg)
5
2
11
9