Luminex bead coupling protocol
We use this process to create our own Luminex assays from standard ELISA reagents. A version of this protocol can be found on protocols.io.
- Activation buffer (100 mM NaH2PO4, pH 6.3)
- Coupling buffer (50 mM HEPES, pH 7.4)
- PBS/1% BSA
- EDC (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide)
- S-NHS (N-hydroxysulfosuccinimide)
Minimize the exposure of EDC and S-NHS to air and moisture. Use fresh aliquots for each coupling reaction and discard after use. Pierce sells individually packaged, small amounts of S-NHS that can be used in a single use fashion.
- 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 for 3 min at 10,000 x g. Carefully 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 for 20 min 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 for 3 min at 10,000 x g, and 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 for 3 min 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.