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Conjugation of Proteins to Carboxylated Fluorescent Microspheres

 Zepto Ultra Fluorescent Microspheres

Introduction


Zepto Ultra Carboxyl Microspheres are designed for surface functionalization with molecules containing primary amines using carbodiimide coupling chemistry using a crosslinkers such as 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC). 

 

The following procedure provides general guidelines for conjugation of antibodies and other proteins to Cytodiagnostics Zepto Ultra Carboxyl Fluorescent Microspheres. Multiple antibodies of different specificities, or other molecules of interest, may be conjugated to beads emitting different fluorescent colors, allowing for multiplexed detection capabilities in a single assay.


Two-Step Conjugation Procedure


A typical conjugation reaction utilizes 10 million Zepto Ultra Carboxyl Microspheres. The following reagents are required and not provided with the product:

  • Activation buffer: 50 mM MES buffer (2-(N-morpholino)ethanesulfonic acid), pH 5.5
  • Washing buffer: 0.1X Phosphate-buffered saline with 0.05% (w/v) Tween 20 (0.1X PBST)
  • Storage buffer: 1% bovine serum albumin in 0.1X PBST, 0.09% sodium azide
  • EDC (Sigma Cat.# E1769): 1 mg/ml in activation buffer (Note: always prepare fresh)
  • IgG or other protein to be conjugated: 0.5 mg/ml in 0.5X PBS

  1. Vortex the bottle/vial containing the beads thoroughly before use to ensure homogeneous suspension of the beads.
  2. Immediately aliquot 10 million beads (10 µL of stock solution as provided) into a 1.5 ml microcentrifuge vial.
  3. Add 50 µL of EDC prepared as described above.
  4. Mix well and activate for 30 min at room temperature.
  5. Add 500 µL of washing buffer prepared according to instructions above and mix well.
  6. Pellet the beads by centrifuging in a microcentrifuge at 500 x g for 5 minutes.
  7. Discard the supernatant and add 50 µL of IgG or other protein to be conjugated.
  8. Mix well and incubate for 4 hours at room temperature with constant rotation.
  9. Add 500 µL of washing buffer and mix well.
  10. Pellet the beads by centrifuging in a microcentrifuge at 500 x g for 5 minutes.
  11. Discard the supernatant and add 500 µL of washing buffer.
  12. Pellet the beads by centrifuging in a microcentrifuge at 500 x g for 5 minutes.
  13. Discard the supernatant and add 250 µL of storage buffer and mix well.
  14. Incubate for 1 hour at room temperature with constant rotation.
  15. The beads are now conjugated to your antibody/protein and ready for your use in your assay. Alternatively, store at 4°C protected from light until use.

* The centrifugation speed for pelleting microspheres is a range between 100 to 10,000 g. A lower/more gentle centrifugation speed with longer time is recommended to form a good microsphere pellet, especially after conjugation. Prolonged centrifugation at high speed may aggregate microspheres irreversibly.


Optimizing the Conjugation Reaction


The concentration ratio of beads to EDC and IgG/protein to activated beads ratio may need to be optimized for optimal conjugation.


Sulfo-N-hydroxysuccinimide (NHS) can also be added with EDC during activation to increase conjugation efficiency. NHS reacts with the o-acrylisourea intermediate formed by EDC and carboxyl group and replaces it with a more stable amine-reactive ester. The molecular ratio between EDC and NHS is typically at 1:1.

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