4. Acquisition

Time requirement: 5 h

Samples: Prepare a ca. 10 mM phosphate buffer solution by dissolving 0.0071 g Na₂HPO₄ in 5 ml D₂O and adjusting the pH*-value to 7.8 using 1 μl of phosphorus acid; add 0.0438 g NaCl to reach a NaCl concentration of 0.15 M. (The pH*-value is defined as the pH-value measured with a pH-meter in a deuterated solution)

NMR tube a: Prepare a 0.02 mM solution of Wheat Germ Agglutinin (Triticum vulgaris) by dissolving 0.7 mg in 1 ml of the buffer solution.

NMR tube b: Prepare a solution which is 1 mM N-Acetyl-D-glucoseamine (0.2 mg/1 ml) and 1 mM D(+)-Raffinose Pentahydrate (0.6 mg/1 ml) in 1 ml buffer solution.

NMR tube c: Dissolve the same amounts of the compounds of tube a and b together in 1 ml buffer solution.

The probe-head should be tuned to the sample. Set and control the temperature to 300 K. Load standard ¹H parameters, adjust the transmitter offset to the residual water signal and record ¹H NMR spectra with presaturation (see Exp. 6.18) of tubes a and b to have reference spectra. Using sample b, determine the 90° selective Gaussian pulse on the decoupler channel using the water signal. For this determination you need phase coherence between the observe and the decoupler channel. Load the STD pulse program and run the STD experiment with tube c. You have to set:

td	32 k
sw	14 ppm
o1	on resonance of H₂O signal
p1	90° selective Gaussian ¹H decoupler pulse [50 ms, 66 dB]
p2	90° ¹H transmitter pulse [8 μs, 5 dB]
d1	20 μs, delay for frequency switching
d2	1 ms, delay within repetition loop for the selective pulses
n	set n to 40 which yields a total irradiation time of 2.04 s
fl	provide a frequency list, which switches the selective pulse between 0 ppm and 40 ppm
ns	4096

As an additional control it is advised to run the STD experiment also on tube b without protein to ensure a zero spectrum under these conditions.

5. Processing

Use standard 1D processing as described in Experiment 3.1.