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  #1  
Unread 05-13-2011, 09:50 PM
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Question Unanswered: CBCANH experment on 700 vnmr

Iam working on protein family which can form dimers (total 20kd size ) using 25mM TRIS , 100 mM KCL Ph-6.8 , protein conc -2mM ( limitaion )

Iam always geting bad quality of CBCANH contures experment on 700 vnmr cryob .

quality of N-H hsqc is good

Could you please suggest me about expermental parameters or buffer conditions to get good quality of CBCANH experment
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Default

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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Default cbcanh

Hi,

Changs the buffer to 50 mM sodium phosphate or sodium acatate and salt concentration to 50 mM NaCl or Kcl. You can try with 1 mM concentrated sample. And do HNCACB insted of CBCANH. It will work. Try with low concentrated (below 1 mM) sample to overcome the problem of dimerization.

cheers, krishna
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