Hello,

I would like to acquire a series of HSQC spectra of an 15N-labeled 12 kDa protein unfolding (over the course of hours) in 3M GuaHCl. I have access to a Varian 600 spectrometer with a conventional probe. Due to the high ionic strength of the sample, tuning and shimming is a little difficult. However, I can record a sort-of-decent HSQC using a WaterGate gradient enhanced WGgNhsqc sequence. I am limited to using a protein concentration of about 250 uM. I've determined the pw90 to be 12.125 at a tpwr of 63.

Using WGgNhsqc, the protein signal is decent (relative to the guanidine), but there is high phase distortion of the guanidine signal at 6.95 ppm. The guanidine signal is "split" half up and half down.

I get slightly different results using a regular gNhsqc -- the protein seems suppressed relative to the guanidine signal, and "antiphase" to it.

I am looking for ways of increasing sensitivity -- I don't mind if the HSQC takes an hour. Does anyone have any practical advise for such an experiment? Is one pulse sequence going to be significantly superior to another in this case?

Hello,

I would like to acquire a series of HSQC spectra an HSQC spectrum of an 15N-labeled 12 kDa protein unfolding (over the course of hours) in 3M GuaHCl. I have access to a Varian 600 spectrometer with a conventional probe. Due to the high ionic strength of the sample, tuning and shimming is a little difficult. However, I can record a sort-of-decent HSQC using a WaterGate gradient enhanced WGgNhsqc sequence. I am limited to using a protein concentration of about 250 uM. I've determined the pw90 to be 12.125 at a tpwr of 63.

Using WGgNhsqc, the protein signal is decent (relative to the guanidine), but there is high phase distortion of the guanidine signal at 6.95 ppm. The guanidine signal is "split" half up and half down.

I get slightly different results using a regular gNhsqc -- the protein seems suppressed relative to the guanidine signal, and "antiphase" to it.

I am looking for ways of increasing sensitivity -- I don't mind if the HSQC takes an hour. Does anyone have any practical advise for such an experiment? Is one pulse sequence going to be significantly superior to another in this case?

Hello,

I would like to acquire an HSQC spectrum of an 15N-labeled 12 kDa protein unfolding (over the course of hours) in 3M GuaHCl. I have access to a Varian 600 spectrometer with a conventional probe. Due to the high ionic strength of the sample, tuning and shimming is a little difficult. However, I can record a sort-of-decent HSQC using a WaterGate gradient enhanced WGgNhsqc sequence. I am limited to using a protein concentration of about 250 uM. I've determined the pw90 to be 12.125 at a tpwr of 63.

Using WGgNhsqc, the protein signal is decent (relative to the guanidine), but there is high phase distortion of the guanidine signal at 6.95 ppm. The guanidine signal is "split" half up and half down.

I get slightly different results using a regular gNhsqc -- the protein seems suppressed relative to the guanidine signal, and "antiphase" to it.

I am looking for ways of increasing sensitivity -- I don't mind if the HSQC takes an hour. Does anyone have any practical advise for such an experiment? Is one pulse sequence going to be significantly superior to another in this case?