Streptavidin Conjugation Protocol

CPNs™ can be readily conjugated to proteins using EDC (N-ethyl-N’-dimethylaminopropyl-carbodiimide), which enables linkage of the protein amine groups (-NH2) to the carboxyl groups on the surface of the CPNs™ (-COOH). Attaching proteins such as antibodies or streptavidin (to enable biotinylated linkage) will create highly selective fluorescent probes, which can target specific molecules of interest. The immense brightness of CPNs™ allows detection of molecules with a high degree of sensitivity. Single molecules can be detected using a wide variety of applications, including flow cytometry, immunocytochemistry and histochemistry methods, and ELISA.

The affinity of targeting molecules varies greatly and an initial titration for the CPNs™:targeting molecule ratio must be undertaken. Similarly, final usage dilution of the CPNs™ in the targeting molecule conjugate will need to be determined empirically.

Protocol

  1. Add 50 μl CPN™ (0.1mg/ml) to 1.5ml tube
  2. Add 1 μl of HEPES 1M
  3. Add 1 μl PEG 8000 5% w/v
  4. Add 3 μl streptavidin (1mg/ml) and vortex (several ratios of CPN™:targeting molecule should be tested to identify the optimum conditions, e.g. 10-100 μl targeting molecule (1mg/ml))
  5. Add 1 μl freshly prepared EDC solution 5 mg/ml
  6. Shake mixture for 4 hours at room temperature
  7. The CPN™:streptavidin conjugates can then be purified from the reaction components by precipitating them using a magnet
  8. Re-suspend in 20 μl sterile Resuspension Buffer (HEPES (20mM), BSA 0.1%, Tween-20 0.02%)

Reagents

  • HEPES (1M), pH 7.4
  • PEG 8000 (5% w/v)
  • Streptavidin (1 mg/ml)
  • EDC [N-(3-Dimethylaminopropyl)-N’-ethylcarbodiimide hydrochloride] 5 mg/ml (Freshly make EDC solution and use immediately. Discard any unused)
  • Bovine Serum Albumin (10% w/v)
  • Tween-20
  • Neodymium magnet