Preparation and Use of Bilirubin Probe

When you receive your order of bilirubin probe, complete the following three steps IMMEDIATELY :

1. FREEZE (at -70°C if possible) THE PROBE
2. FREEZE (at -20°C) STANDARD(S)
3. REFRIGERATE (at 4°C) STORAGE AND MEASURING BUFFERS

Preparation of stock solution for the Probe:

Add storage buffer to the vial of probe powder to give a final concentration of ~100 µM. The approximate molecular weight of the probe is 15000 g/mole; therefore, for every 200 µg probe, add 133 µL storage buffer and for 1 mg probe, add 667 µL. Once storage buffer is added to the probe, store at 4°C. The storage buffer consists of 20 mM HEPES, 140 mM NaCl, 5 mM KCL, 1 mM Na2HPO4, 1 mM EDTA and 0.05% sodium azide. The pH at room temperature is 7.4± 0.1.

Measuring buffer:

The measuring buffer consists of 20 mM HEPES, 140 mM NaCl, 5 mM KCl, and 1 mM Na2HPO4. The pH at room temperature equals 7.4 ± 0.1. Store the measuring buffer at 4ºC. The recommended concentration of each probe to be used in the measuring buffer is approximately 0.5 µM. The concentration can be increased or decreased depending on the efficiency of the fluorometer used.

Cuvettes:

Cuvettes made of glass or quartz are recommended for a measurement using one of the probes. These cuvettes must be very clean and all traces of soap rinsed away. A final rinse of ethanol and then drying under a nitrogen stream is recommended. Disposable cuvettes can also be used. Plastic cuvettes made of polystyrene from Sarstedt (cat# 67.741) have been found to work well with our probes. However acrylic cuvettes can leach a substance that reacts with the probe. Cuvettes made of other materials can be easily tested by determining if the probe ratio changes over time.