Maybe this is all given, but just in case...
- try NUS ;-) , no useful enhancements for typical 3D NMR of proteins,
but it will help get decent resolution on tough constructs
- protein size factors in; should be able to do quite a bit with a 10kDa
protein at 40 or 50 uM in 5mm cryoprobe at 800 MHz, for example, but 50 kDa
another story
- they need to test to see if protein tolerates higher T; narrower lines at
30C or 37C can be a considerable help; high T may worsen aggregation or may
not;
- they need to do stability tests to see if the protein can tolerate
reduced salts (Pi, NaCl, Tris, etc)
- see note below, but has the group done any construct optimization? If
this is their 'first try' they have to keep working on it (salts, buffers,
glycerol, detergents, also trim unstructured amino acids, etc)
- they need to do stability tests (gels, CD, hsqcs) to see if there is the
luxury to burn spectrometer time to help out
- aside: large volume 5mm shigemi's are a mixed bag in my experience; they
technically work, but the boost is modest (~10%) and they are more work to
shim, will exacerbate Q issues, and if you end up with slightly broader
lines, you've lost the benefit,so I would not try it here, but mentioning
it for completeness
- if salt is high, a 4mm tube can give better performance
Maybe I'm off base with this, but just in case: biochem groups are skilled
at preparing 5-20 uM proteins, which is hard enough already, but getting to
NMR accessible concentrations is another task altogether. If this is not
an NMR lab, then it's great they are interested, but they may need coaching
- a year or two of construct optimization is a normal part of many PhD
theses in NMR structbio, especially since many "easy, soluble" proteins
have been done already leaving more challenging proteins to be studied -
so depending on their tolerance for that investment of time/resources,
that's what they may need to consider.
DSR
On Tue, Oct 29, 2024 at 9:12 AM Anklin, Clemens via groups.io wrote:
> Dear Charlie,
>
> the only large diameter cryoprobes we have built are all X-observe. We
> have 10mm 13C/1H dual probes and for very high fileds we have built some 8
> mm TXO probes that are optimized for 13C or 15N. 8 and 10 mm probes will
> suffer greatly from almost any level of salt concentration.
>
> I do remember the time back in the 90's when we built 8 mm conventional
> HCN probes only to be disappointed by their performance. They took about 3
> times the volume and as soon as there was salt or buffer in the solution
> you were lucky to get 60% more signal than the 5mm probe.
>
> The problem is only going to get worse with cryoprobes or at higher
> fields.
>
> Sorry for the bad news but there is no magic bullet here.
>
> Regards
>
> Clemens
>
>
> On 10/28/2024 14:34, Charles Fry via groups.io wrote:
>
>
>
> Hi all,
>
>
>
> I’m supporting a protein research group that needs to work at low
> concentrations (20-50 uM) and is asking about using a 10mm cryoprobe. I’ve
> talked about line shape and shimming on such probes, and also mentioned 8mm
> probes. I’m uncertain about how bad Q-spoiling by buffers might be. I’ve
> used X-direct-detect 10mm probes, but have only talked with vendors about
> 8mm probes and those discussions were at least 10 yrs ago. What I recall is
> that 5mm probes are better optimized for both line shape and sensitivity,
> and that overcomes (at least most of) the advantages of larger sampling
> volume with the bigger diameter probes. But I’ve always wondered how much
> the economics of such expensive devices -- a larger diameter probe having
> considerably narrower utility -- might influence such discussions. One
> aspect is whether new shimming technologies might make the larger diameter
> probes more competitive than they used to be?
>
> The research group remains interested in the possibilities. I agreed to
> ask in this forum who might have such probes, especially those that would
> be open to outside academic use. Even if you have a probe that is not
> available for use to others, it would be great to hear more about their
> utility for 1H- and 13C-detected 2D and 3D experiments.
>
>
>
> Cheers,
>
> Charlie
>
>
>
>
>
>
> -- ========================
> ==========================
> ======================
> Clemens Anklin Ph.D. Vice President Bruker BioSpin NMR Applications &
> Training 15 Fortune Drive NEW Phone: 978-313-5NMR(5667) Billerica MA 01821
> twitter: _at_canklin web: http://www.bruker.com
> e-mail: clemens.anklin_at_bruker.com
> =========================
> ==========================
> =====================
>
>
>
--
---------------------------------------------------------------------------
David Rovnyak, Professor of Chemistry, Bucknell Univ., Lewisburg, PA 17837
Twitter : NMR_Guy <http://twitter.com/NMR_Guy
Research: http://www.facstaff.bucknell.edu/drovnyak/
Bucknell Chemistry: http://www.departments.bucknell.edu/chemistry/
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