View source for Collecting a 15N Edited HSQC

Collecting a Superb 15N edited HSQC (NHSQC)

#	Concentrate your protein as much as possible. It should be at least 50uM for 2D work and around 1mM for 3D work.
#	Spike in 10% D2O.
#	Place in a Shigemi tube (if low volume) or in any other NMR tube. Cap and seal.
#	Insert your sample into the spectrometer. Create a new dataset.
#	Open experimental parameter set ‘NHSQC’. If you can’t find it, open hsqcetf3gpsi. Also, double check that this is the pulse program.
#	Edit the number of points in the direct and indirect dimension. Should be 2k and >= 128. 
#	The number of dummy scan should be at least 16.
#	The number of scans should be adjusted depending on the concentration. For 1mM protein, 2 scans is ok. For 500uM protein, 8 scans will be necessary.
#	Edit your sweep width in the indirect dimension. A larger sweep width will prevent folding of the spectrum but will decrease digital resolution.
#	Edit your 15N center position. It should be between 115-120 ppm.
#	Edit the ZGOPTNS. Add –DLABEL_CN if the protein is double labeled. This will tell the pulse program to decouple 13C.
#	Check experiment time with ‘expt’.
#	Lock ‘lock h2o+d2o’.
#	Tune ‘atma’.
#	Shim ‘topshim’ or ‘topshim 3d’.
#	Find p1 ‘pulsecal’.
#	Calculate receiver gain ‘rga’.
#	Zero go ‘zg’.
#	Process the specrum with ‘xfb’.
#	Edit phasing manually with ‘ph’.
#	Rule of thumb - If you can collect an amazing NHSQC in 10 min you are ready for 3D work.

[[category:Data_Collection]]
[[category:NMR_Usage]]