Luminex Bead Coupling
Vortex the bead stock suspension to yield a homogeneous bead suspension .
Dissolve approximately 10 mg each of EDC and S - NHS into 2 microcentrifuge tubes and resuspend in deionized water at 50 mg / mL .
Centrifuge the 100 μl bead suspension at 10,000 x g .
Carefully remove and discard the supernatant .
Resuspend the beads in 80 μl activation buffer .
Add 10 μl of S - NHS solution and 10 μl of EDC solution to the bead suspension .
Incubate with agitation at room temperature in the dark at roughly 900 rpm .
Dilute your protein stock solution with coupling buffer to a concentration of 0.1 mg / ml in a volume of 100 μl .
Optimal coupling may occur at a concentration within 25 - 250 μg / ml .
Note the protein stock cannot have any other amine groups present .
Centrifuge the beads at 10,000 x g .
Carefully remove and discard the supernatant .
Add the diluted protein solution .
Agitate the tube with activated beads and protein solution overnight at 4C at roughly 900 rpm in the dark ( wrapped in foil ) .
Centrifuge the beads at 10,000 x g .
Discard supernatant .
Wash the beads three times with PBS / 1 % BSA .
Resuspend the bead pellet in 1 ml PBS / 1 % BSA .
Determine bead concentration using Luminex and adjust amount of stock used accordingly .
