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Inject known standards of Cy3 NHS-ester and edaATP at the same concentration as the reaction mixture to identify peaks.

Distil triethylamine (500 ml), discarding the first and last 10% of the distillate. Use the middle 80% of the distillate (see Note ).

Add cold (4°C), distilled, deionized water to 139.4 ml distilled triethylamine to give 1 L of a 1 M solution (see Note ).

In a fume hood, put dry ice in the Buchner flask, and with the solution in ice, bubble CO2 through the solution until the pH is 7.5–7.6 (approximately 2 h) using the scintered glass bubbler. Keep the Buchner flask, containing the dry ice, raised above the solution to reduce the risk of sucking back.

Store triethylammonium bicarbonate (TEAB) at 4°C in a well-stoppered container. It lasts approximately 1–2 months, but the pH gradually rises with time. In this case, rebubble CO2 through it.

Preequilibrate the DEAE-cellulose column with 10 mM TEAB, pH 7.6 at 1 ml/min at 4°C.

Alter the pH of the reaction mixture to 7.6 using acid or base, reduce the conductivity by dilution in water so it is close to that of 10 mM TEAB and load onto the column.

Wash the column with 10 mM TEAB, pH 7.6 at a flow rate of 1 ml/min until no more pink material is eluted.

Elute the nucleotide with a linear gradient of 10–800 mM TEAB (total volume 600 ml). Follow the absorbance at 254 nm. Unreacted edaATP is eluted first followed by Cy3-edaATP (see Note ).

Identify the fractions containing Cy3-edaATP by measuring the absorbance at 550 nm and 260 nm.

Pool fractions containing Cy3-edaATP and remove TEAB by rotary evaporation. Use a flask with a capacity at least four times the volume of the solution.

Fill the condenser with dry ice–isopropanol.

Add the pooled fractions to the flask, rotate, and slowly apply the vacuum to begin evaporation. Warm the flask in a water bath at 30°C. Reduce the volume to ∼5 ml. When the solution ­volume is reduced to 10–20%, frothing may begin (see Note ).

Add methanol (∼10% of initial solution volume) and repeat the evaporation.

Repeat methanol additions and evaporation three times: ­during this it should be possible to remove essentially all the solvent before adding more methanol. At the final stage, evaporate all of the methanol. The Cy3-edaATP will remain as a gum.

Dissolve in <3 ml methanol and transfer to a pear flask (10 ml) and reconcentrate, with very careful application of the vacuum to avoid frothing. Finally, dissolve in water or buffer and adjust to pH  ∼  6–7 before storing at −80°C (see Note ).

Measure the absorbance spectra of Cy3-edaATP in 50 mM Tris–HCl, pH 7.5 between 220 and 700 nm. Taking the extinction coefficient for the Cy3 to be 150,000 M−1 cm−1 at 552 nm () and the extinction coefficient for adenosine to be 15,200 M−1 cm−1 at 260 nm, calculate the concentration of the nucleotide (see Note ).

Characterize Cy3-edaATP by HPLC using the same method as above. The major peak should be Cy3-edaATP. Check for the presence of Cy3-edaADP, edaATP, and edaADP. Determine the purity by integrating the Cy3-edaATP peak with any other peaks (see Note ).

Measure the fluorescence excitation and emission spectrum of Cy3-edaATP in 50 mM Tris–HCl (pH 7.5). Typically, 1 μM in a solution of 60 μl will be used. Use the peak wavelength from the absorbance measurement as the excitation wavelength to measure the emission. Then, use the peak in the emission spectrum for the excitation spectrum. Add an excess of the protein of interest to the sample (e.g., 10-fold) and repeat the measurement (see Notes 14 and 15). Compare the two spectra to determine the change in fluorescence when bound to protein.