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Unread 09-29-2011, 02:12 PM
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Many backbone 3D experiments start to fail when your protein reaches 20+ kDa molecular weight.

The CBCANH experiment is also one of the most insensitive experiments out there. When you combine all of the above, it's not surprising that the experiment doesn't work too well at all.

There are several solutions around this:

1) You can run a HNCACB experiment, which tends to have significantly better sensitivity, and gives you the information you need.

2) You can rely on a HNCA experiment combined with the CBCA(CO)NH experiment to give you a lot of the same information

3) Check the water suppression method that you're using. While I cannot give you a direct answer when it comes to exactly what you should use (I use Bruker machines), I found that the "default" Sinc1.1000 shaped pulse used in the water flipback pulses didn't work nearly as well as some of the other choices, such as the square pulses or the ramped square pulses.

When I used the Sinc1 pulses, the water suppression was poor, and a lot of my backbone experiments would look like there was very little signal. Using more robust water suppression helped restore a lot of the peaks.
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