Preparation and Use of ADIFAB
When you receive your order of ADIFAB, complete the following three steps IMMEDIATELY :
1. FREEZE (at -70°C if possible) LYOPHOLIZED
Preparation of ADIFAB stock solution:
Add storage buffer to the vial of lyophilized ADIFAB powder to give a final concentration of ~100 µM. The approximate molecular weight of ADIFAB is 15000 g/mole; therefore, for every 200 µg ADIFAB, add 133 µL storage buffer and for 1 mg ADIFAB, add 667 µL. Once storage buffer is added to ADIFAB, store at 4°C. The storage buffer consists of 50 mM TRIS, 1 mM EDTA, and 0.05% sodium azide. The pH at room temperature is 8.0 ± 0.1.
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 ADIFAB to be used in the measuring buffer is approximately 0.2 µM. The concentration can be increased or decreased depending on the efficiency of the fluorometer used.
Cuvettes made of glass or quartz are recommended
for the measurement of ADIFAB. 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# 67.754) have
been found to work well with the ADIFAB2 probe. However acrylic cuvettes
have been found to leach a substance that reacts with the probe. Cuvettes
made of other materials can be easily tested by determining if the ADIFAB
ratio changes over time.