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tRNA Probes

Fluorescent aa–tRNAs

The emission intensity, wavelength, and lifetime of the NBD dye are very sensitive to the presence of water.  Because of its small size (two rings), NBD has a minimal effect on sample interactions. In fact, εNBD-Lys is incorporated into protein at the same rate and to the same extent as unmodified Lys by the ribosome during in vitro translation.  Among its many potential uses, NBD can distinguish between nonpolar and aqueous environments, detect conformational changes that alter probe exposure to the environment, and detect association of the NBD-labeled protein with another protein, an RNA or DNA, a small molecule, and/or a multicomponent assembly.  NBD can also act as either a donor or an acceptor in FRET experiments, as well as a probe of accessibility or compartmentalization by using hydrophilic or hydrophobic collisional quenchers of fluorescence.  In addition, the kinetics and thermodynamics of an interaction or process can be characterized and quantified by NBD spectral changes.  NBD = 7- nitrobenz-2-oxa-1,3-diazole-; λex ~ 466 nm; λem ~ 535 nm, but is solvent-dependent.

Fluorescein excitation (absorption), and hence its emission intensity, is very sensitive to pH and the local electrostatic microenvironment.  Fluorescein (λex ~ 490 nm; λem ~ 520 nm) positioned in the inside corner of the tRNA L is particularly useful in monitoring conformational changes elicited by, for example, protein binding to an aa-tRNA that alters phosphate proximity to fluorescein.  Fluorescein is also an excellent FRET donor or acceptor in ribosomal or other complexes.  However, ε fluorescein-Lys or fluorescein-S-Cys cannot be incorporated into protein in vitro using a modified aa-tRNA because the dye is too large (4 rings).

Many other dyes have been covalently attached to Lys-tRNALys, Cys-tRNACys, or Lys-tRNAamb, some of which must be supplied by the customer to avoid trademark complications. If you are interested in using a particular dye or pair of dyes, please consult our custom synthesis options.  

Each tube contains 400 pmoles of aa-tRNA that have been dialyzed into 25-30 μl of 1 mM KOAc (pH 5.0), 2 mM Mg(OAc)2.
 
Item #

Fluorescent  aa-tRNAs

 Quantity per tube

Cost per tube

A-15

εNBD-[14C]Lys-tRNAamb

400 pmoles

$ 125
       
A-55

non-RA  εNBD-Lys-tRNAamb

400 pmoles $ 125
 
       
C-55

non-RA E. coli  NBD-S-Cys-tRNACys

400 pmoles $ 130
 
C-35

yeast  NBD-S-[14C]Cys-tRNACys

400 pmoles

$ 130
C-65 non-RA  yeast  NBD-S-Cys-tRNACys 400 pmoles $ 130
 
L15

E. coli  εNBD-[14C]Lys-tRNALys

400 pmoles

$ 125
       
L-55

non-RA E. coli  εNBD-Lys-tRNALys

400 pmoles $ 125
 
L-35

yeast  εNBD-[14C]Lys-tRNALys

400 pmoles

$ 130
       
L-65

non-RA  yeast  εNBD-Lys-tRNALys

400 pmoles $ 130