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Preparation and Use of PAL201 and OAL1301
When you receive your order of probes, complete the following three steps IMMEDIATELY : 1. FREEZE (at -70°C if possible) THE PROBES Preparation of stock solution for the Probes: Add storage buffer to the vial of probes powder to give a final concentration of ~100 µM. The approximate molecular weight of each 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 a 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. Assay Method: You can determine unbound free fatty acid levels in an aqueous solution with a simple, one step procedure: add a small amount of the reagent to a solution suspected of containing unbound free fatty acid and measure the resulting fluorescence. The excitation wavelength for the probes is 375 nm and the emission wavelengths are 550 and 457 nm. Characteristics
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