Fluorescent probe medium, preparation

Texas Red hydrazide is soluble in DMF and may be dissolved as a concentrated stock solution in this solvent prior to the addition of a small aliquot to an aqueous reaction medium. The solid and all solutions made from it must be protected from light to avoid photo-decomposition. Prepare the stock solution fresh immediately before use. A suggested protocol on the use of this fluorescent probe may be obtained by following the method outlined for fluorescein-5-thi-osemicarbazide in Section 1 of this chapter. Optimization may be necessary to achieve the best level of fluorescent modification (F/P ratio) for a particular application. 

The majority of fluorescent probes are water-insoluble and must be dissolved in an organic solvent prior to addition to an aqueous reaction medium containing the DNA to be labeled. Suitable solvents are identified for each fluorophore, but mainly DMF or DMSO are used to prepare a stock solution. Some protocols utilize acetone when labeling DNA. However, avoid the use of DMSO for sulfonyl chloride compounds, as this group reacts with the solvent. For oligonucleotide labeling, the amount of solvent added to the reaction mixture should not exceed more than 20% (although at least one protocol calls for a 50% acetone addition—Nicolas etal., 1992). 

Many of the crystals were prepared in the presence of azide, which is known to enhance the MgADP inhibition [17], and azide has been resolved in a crystal [38]. Thus, the mutually similar structures, including that of EFi [39] which was also prepared in the presence of azide, must all represent, basically, the MgADP-inhibited state. In this state, 7 is orientated at 80° [18], as in the 80° intermediate during active rotation. Fluorescence energy transfer between probes on a / and rotating 7 has indicated that, of the two active rotation intermediates at 0° and 80°, the crystal structures are much closer to the 80° intermediate [40]. At 80°, we expect one catalytic site to be open to the medium (asterisks in Fig. 14.2B), which is consistent with the idea that the crystal structures with a fully open catalytic site mimic the 80° state rather than the ATP-waiting state. 

Pyrene is a useful probe for studying the polarity of the medium because its fluorescence vibronic structure is very sensitive to the microenvironment [92,93]. In particular, the ratio of the intensities of the first (373 nm) to the third (384 nm) fluorescence vibronic maxima (R = I/III) strongly decreases with the decreasing polarity of the medium. The interaction of pyrene (13) with the three CDs has been thoroughly investigated by photophysical methods. There are considerable discrepancies among the results of the various reports, which are probably related to the very low solubility of 13 in H2O (<5 X 10 M). The low solubility imposes the need for special precautions when preparing the samples [94]. 

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