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.
- Add 50 μl CPN™ (0.1mg/ml) to 1.5ml tube
- Add 1 μl of HEPES 1M
- Add 1 μl PEG 8000 5% w/v
- 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))
- Add 1 μl freshly prepared EDC solution 5 mg/ml
- Shake mixture for 4 hours at room temperature
- The CPN™:streptavidin conjugates can then be purified from the reaction components by precipitating them using a magnet
- Re-suspend in 20 μl sterile Resuspension Buffer (HEPES (20mM), BSA 0.1%, Tween-20 0.02%)
- 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)
- Neodymium magnet