Quenching molecular probes

Ricin A-chain (Inland Labs) at 3 mg/mL in PBS was reduced for 30 min with 30 mM DTT at 30°C. Excess DTT was removed by Sephadex G-25 gel filtration in 5 mM sodium acetate buffer, pH 4.7, containing 50 mM NaCl and 0.5 mM EDTA. The thiol content was assessed by Ellman s assay (Ellman, 1959). MIANS (Molecular Probes) was added at a ratio of 0.9 mole MIANS 1.0 mole thiol. The reaction mixture was incubated at ambient temperature for 15 min after the pH was raised to 7.0 using 1 M Tris.HCl buffer, pH 7.4. The MIANS was quenched by addition of 1 mole equivalent of freshly prepared cysteine.HCl/mole MIANS and incubation for an additional 15 min. Any remaining thiol groups were alkylated by adding a 5-fold molar excess of iodoaceteimide over total thiol and after an additional 30 min, the protein (MIANS-ricin A-chain) was dialyzed against 0.1 M Tris.HCl buffer, pH 8.5. 

The termination reaction of free radical polymerization is a typical example of an intermacromolecular diffusion controlled reaction.3 Photophysical studies carried out in the 1980 s demonstrated for the first time that the reaction is solvent- and molecular weight-dependent. The experiments involved triplet quenching of probes attached to polymer chain ends. A benzil group was linked to the end of one PS sample (PS-B) and an anthryl group was linked to the end of a second PS sample (PS-A). The quenching rate coefficient kq of the benzil phosphorescence by anthryl groups is given by Eq. (3.26), where r0 is the lifetime of benzil phosphorescence in the absence of anthryl and ris the benzil phosphorescence lifetime in the presence of anthryl in concentration [A],... 

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