[nmrlearner]

Originally posted by alan.robot@Sep 14 2005, 09:19 PM
Yes, I read the same paper and tried it with favorable results.* I am working on a ~10kDa RNA binding protein from Drosophila which would precipitate after only 3 days in the NMR.** After optimizing the buffer conditions it would last ~ a week but still slowly precipitate.* I tried 50mM Arg/Glu and now my sample is happy for several weeks! Still have really bad s/n because of all the salt, though (if I go lower than 100mM KCl it aggregates, and the Arg-HCl and K+ Glu salts are really driving up the effective salt concentration). All that and parts of protein seem to be exchange-broadened* ** But at least it lasts long enough to do most triple resonance experiments now.
Cheers,
Alan

P.S.* Like the paper says it's important to add the arg/glu when the sample is fairly dilute, that is before the final concentration.* Also, prepare some D2O with 50mM arg/glu to avoid cloudiness when adding the 10% to your sample.

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Thanks for your feedback, Alan! It is nice to know that this method works not only for the authors.

Still have really bad s/n because of all the salt, though (if I go lower than 100mM KCl it aggregates, and the Arg-HCl and K+ Glu salts are really driving up the effective salt concentration).

Does the protein precipitate with or without Arg-HCl and K+ Glu when you go below 100 mM KCl? If without, may be 50 mM Arg-HCl and K+ Glu can replace 50 mM KCl?

All that and parts of protein seem to be exchange-broadened* **

Well.. sounds like an RNA binding protein. You may want to try to change the exchange behavior by going to a different field-strength or/and to use CPMG in HNCA and other backbone assignment experiments.

Mark