Complexing Probes

Kolubayev T, Geacintov N E, Paillotin G and Breton J 1985 Domain sizes in chloroplasts and chlorophyll-protein complexes probed by fluorescence yield quenching induced by singlet-triplet exciton annihilation Biochimica Biophys. Acta 808 66-76... 

Platinum complexes probes of polynucleotide structure and antitumour drugs. S. J. Lippard, Acc. Chem. Res., 1978,11, 211-217 (56). 

T. P. Frandsen, B. B. Stoffer, M. M. Palcic, S. Hof, and B. Svensson, Structure and energetics of the glucoamylase-isomaltose transition state complex probed by using modeling and deoxygenated substrates coupled with site-directed mutagenesis, J. Mol. Biol., 263 (1996) 79-89. 

J. Screen, E. C. Stanca-Kaposta, D. P. Gamblin, B. Liu, N. A. Macleod, L. C. Snoek, B. G. Davis, and J. P. Simons, IR-spectral signatures of aromatic-sugar complexes Probing carbohydrate-protein interactions, Angew. Chem. Int. Ed., 46 (2007) 3644-3648. 

Dickmeis, T., P. Aanstad, M. Clark, N. Fischer, R. Herwig, P. Mourrain, P. Blader, F. Rosa, H. Lehrach and U. Strahle. Identification of nodal signaling targets by array analysis of induced complex probes. Dev. Dyn. 222 571-580, 2001. 

Barton, J. K.,Tris (phenanthroline) metal complexes probes for DNA helicity,... 

A similar unique effect of PMA on the photophysics of RuCbpy) is observed at pH 5, for example both the lifetime and luminescence intensity of RuCbpyjj show maxima at pH of about 5. The luminescence of the probe also exhibits a blue spectral shift at this particular pH compared to other pH. The change in the photophysical properties are due to binding of RuCbpyjj " " into a partially coiled or swollen polymer PMA at pH 5. The binding is electrostatic in nature and the ligands of the organometallio complex probe are quite restricted in a hydrophobic environment, so that unlike more mobile systems such as water or a stretched polymer, complete relaxation of the excited state is not achieved. Hence, the lifetime and the yield of luminescence increase accordingly and the emission spectra show a blue shift.(42)... 

At least two probes are required, the test probe and the control probe. The test probe can be derived from either mRNA or total RNA. The control probe can either be a complex probe derived from RNA of a control cell type or a probe that will hybridize to all target DNA such as a vector probe. If a complex control probe is used, then levels of expression can be directly compared between two complex probes and small differences between samples can be detected. The quantity of complex probe needed will be governed by the complexity of the RNA, and in general, the larger the quantity of RNA used the better the probe. For human RNA 50-100 p,g total RNA or 1-2 qg polyA+ mRNA can be used. 

Fig. 2. Resultant scan from a cDNA microarray analysis of Stat3-specific gene targets in mammary epithelial cells. (A) Portion of a 15,000 mouse cDNA array, hybridized with Cy3 (inactive Stat3) and Cy5 (active Stat3)-labeled cDNA probes from a mouse mammary epithelial cell line. Note the range of hybridization intensities between cDNAs spots on the array, representing relative differences in the expression of these transcripts in the complex probe populations. (B) and (C) Differential hybridization to individual cDNAs on the same part of the array is evident in separate scans of Cy-3 (B) and Cy-5 (C) labeled probes. These cDNA clones therefore represent likely gene targets of the Stat3 transcription factor.

Akimoto S, Takaichi S, Ogata T, Nishinura Y, Yamazaki I and Mimuro M (1996) Excitation transferin carotenoid-chlorophyll protein complexes probed by femtosecond fluorescence decays Chem Phys Lett 260 147-152... 

V. PROBING MORE COMPLEX STRUCTURES AND MORE COMPLEX PROBES... 

The excitation efficiency of bound and free ground state probe molecules has to be known in order to perform quantitative measurements. The easiest experimental approach is to work at an isosbestic point, so that the excitation efficiencies are equal. Unfortunately, this is not always possible when lasers are employed. When a system is being excited at a wavelength different from the isosbestic point, the ratio of free and complexed probe in the excited state will be different from that encountered in the ground state prior to excitation. The ratio in the excited state can be determined by taking into account the molar absorptivities of the free and complexed ground state probe at the excitation wavelength. 

Figure 1 Ruthenium carbene complexes probed for metathesis activity in the gas phase.

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