I'm planning to do a 15N-1H HSQC experiment to verify the formation of an alleged DNA-Protein complex.
I've found a protocol in which the concentration of the salts in the buffer are roughly of -500mM of NaCl,
-50mM HEPES,
-1mM DTT,
-50mM TRIS,
-0.1mM EDTA.
I'm wondering at what extent these salt concentrations could affect the nmr experiment (e.g. pulse duration, quality of signal, matching, tuning....)
First of all there is a high influence on tuning and matching of your solvent deuterium signal. Probeheads that are not dedicated to protein work may be not able to be tuned at all (due to narrow filter width on 1H)... Secondly high concentrations could result in drastically longer 90° pulse durations with all effects concerning heat load, dephasing etc. All these effects combined could lead to lower S/N in your experiment and longer necessary duty cycles and thus to longer experimental times. Nevertheless, you are not the first to measure highly saline NMR samples and there is a solution for nearly every challenge! Good luck...
Yes, salt can be a problem. It makes tuning and matching the probe difficult (especially the matching) and it will increase the length of a proton 90 degree pulse. You are looking at a protein/DNA complex and using a 15N channel as well so chances are the probe may be ok with 500 mM NaCl if it is a room temperature probe. If it is a cryoprobe then 500 mM will be a big problem.
There are some options that are still possible but they depend on hardware. For example Varian/Agilent make salt tolerant probes that accommodate two 2.5 mm tubes instead of the standard one 5 mm tubes. This all depends on the magnet hardware you have access too.
I suggest you just try it at 500 mM NaCl and see if the probe can handle it. I understand you don't want to drop the salt level because you need to show the binding is not just driven by general nonspecific ionic interactions.
Good luck!
Hi
Which type of protocol did you find ? Was it just for the protein purification or was this found to be necessary for the stabilization of your protein-DNA complex ? Biochemists usually do protein purification in presence of 0.5 M salt - but this is not always necessary. May be you can check using gelfiltration whether such a high salt concentration is really necessary to prevent your protein from precipitation. Do you have other methods to check whether your protein-DNA complex is formed ? Then try to check whether salt concentration can be diminished.
Beate
Dear Antonio,
speaking for a 600 MHz cryoprobe I can say the following:
1) If you are using a 5 mm probe your sensitivity will be reduced by roughly a factor of two compared to low salt condtions.
2) You can always use a 3 mm tube in a 5mm probe. In this case you will hardly have any loss, because not only the salt concentration, but also the overall amount of salt and the cross section of the tube is of importance (see Scott Robsons answer).
This means that at high salt you will have 20-30% less sensitivity from the 160 ul in the 3mm tube compared to the 550 ul in the 5mm tube (assuming identical protein conc. in both tubes!). We (and many others) often use this approach.
If possible you should dissolve the total amount of protein you have in the 160 ul of a 3mm tube - than you will hardly loose any sensitivity! This of course works only if the solubility of the protein is not preventive....
3mm tubes in 5mm probheads offer in addition advantages in respect to line-shape and related to this solvent suppression!
Hope this helps!
Cheers
Alfred
P.S. For non-cryo probes the effect is by far less dramatic, as these probes have lower Q values!
One point I would add. If your 90° pulse is reduced by a factor of two, the signal you get is reduced by nearly a factor of two, althouch you adjusted the 90° pulse to maximum signal. Because the absorption of the salt solution works when the magnetic frequency ist emited. I found the percentage of absportion depence also on the frequency. What you see for proton exitation isn't as much for 13C or 15N. So I often must adjust the proton decoupling in salt solution by a factor of two to reach 90° but the 13C is reduced only 10%. For high salt konzentrations all signal is lost in the sample. You can see it nealy on the hight of the wobble signal, this is aslo influened.
All of the above is good advise. Also you might want to check out "Low-Conductivity Buffers for High-Sensitivity NMR Measurements" by Alexander E. Kelly et al. JACS Articles on the Internet. Also, "NMR_sample_preparation-Structural Biology Facility", structuralbiology.uchc.edu/protocols/pdfs/nmr_sample_preparation.pdf. Also www.tandfonline.com/doi/pdf/10.1080/108260794080113621 on salt concentration effects. Good luck in your research!
Neither Tris nor Hepes will not affect on N15 HSQC, because protons of these buffers are outside amide ppm range. I think that even NH2 proton of TRIS will not be problem. So you dont need to have deuterated buffers. 50 mM is OK for maximal reciever gain.
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