Preparation of NMR Buffers Protocol

By: Pharma Tips | Views: 2178 | Date: 17-Aug-2013

The preparation of a biomolecule for NMR studies usually requires exchanging the sample in an aqueous solution appropriate for conducting the proposed NMR studies. The two main requirements that must be met to successfully acheive this include:

Preparation of NMR Buffers Protocol

NMR spectroscopy

3. Preparation of NMR Buffers Protocol:
3.1.Stock Solutions:
3.1.1  0.1M stock solution of Na2HPO4.
        Weigh 141.96g of Na2HPO4 (anhydrous) and dissolve to 1000 ml (one Liter)     with ddH2O.
3.1.2  0.1M stock solution of NaH2PO4.
           Weigh 119.98g of NaH2PO4 (anhydrous) and dissolve to 1000 ml (one Liter) with ddH2O.
NB: The weight of each salt needed to prepare one liter of a 1M solution varies, depending on the hydrated state of the salt.
3.1.3    .50m M stock solution of DSS.
           Weigh 1.0915g of DSS and dissolve to 100 ml with ddH2O.
3.1.4. NaOH solution.
3.1.5. HCl solution.
3.2. NMR Buffer number 1:
3.2.1. Measure 28.85 ml of the 1M stock solution of Na2HPO4 and 21.15 ml of the      1M stock solution of NaH2PO4 into a 1000 ml graduated cylinder.
3.2.2. Mix and add ddH2O to 850 ml.
3.2.3. Add 100 ml of D2O.
3.2.4. Add 10 ml of the 50 mM DSS stock solution.
3.2.5. Mix well and adjust to pH to 7.0 (if necessary) with HCL or NaOH solution.
3.2.6. Make volume up to 1000 ml with more ddH2O
3.2.7. Store in a labeled, dated bottle in the refrigerator.
3.2.8. Check for impurities before use.

3.3.NMR Buffer number 2:
3.3.1. Measure 28.85 ml of the 1M stock solution of Na2HPO4 and 21.15 ml of the    1M stock solution of NaH2PO4 into a 1000 ml graduated cylinder.
3.3.2. Mix and add ddH2O to 980 ml.
3.3.3. Mix well and adjust to pH to 7.0 (if necessary) with HCL or NaOH solution.
3.3.4. Make volume up to 1000 ml with more ddH2O
3.3.5. Store in a labeled, dated bottle in the refrigerator.
3.3.6.  Check for impurities before use.



Buffer Exchange Procedure for the Preparation of NMR Samples

The preparation of a biomolecule for NMR studies usually requires exchanging the sample in an aqueous solution appropriate for conducting the proposed NMR studies. The two mainrequirements that must be met to successfully acheive this include:

1. Useage of buffer components lacking covalently attached protons. This is important as the buffer will typically be present at much higher concentrations than that of the biomolecule, thus causing the NMR signals of the buffer to interfere with those of the biomolecule.

2. Inclusion of deuterated water (D2O) at a level of 5 % or greater. This is necessary as the stability of the NMR spectrometer is maintained by continuously monitoring the deuterium signal from the solvent.

There are two other important points to keep in mind. The first is that the total ionic strength should be kept as low as possible (compatible with the solubility/stability of your particular biomolecule). This is important as the coupling of the RF signal in the coil to the sample will be more efficient as the ionic strength is lowered. Thesecond is that the pH should be kept low (below 6.5) if possible. This is because the exchange of the backbone amide proton is base-catalyzed; thus its exchange rate increases as the pH is raised. This can be bad because if the exchange rate becomes too rapid, the amide proton will exchange during the course of the nmr pulse sequence, and therefore the amide group will not be detected.

Although there are many ways to prepare samples of biomolecules in aqueous solutions containing 5% D2O, the following is one that I have been found to be particularly simple, reliable, and fast!
1. Transfer your biomolecule to standard dialysis tubing and dialyze against your final desired NMR buffer (e.g. 25 mM dibasic sodium phosphate, 25 mM NaCl, 5% D2O, pH 6.0 (as a guide to figure out the number of times to dialyze and the volume, simply calculate the cumulative effective dilution factor necessaryto bring down any contaminating species to 0.1 times the protein concentration).

2. Wash a centricon unit (these are one-time centrifugal concentrators sold by Millipore; various molecularweight cutoff membranes are available, e.g. 3k, 10k, 30k, ..) using an aliquot of your nmr buffer (rinse once, discard, refill, and then centrifuge for 5 mins). Transfer your protein solution and concentrate to about 150 - 200 uL.Importantly, use the cap supplied, not parafilm, as the parafilm will create an airtight seal; this can be a problem as a chilled centrifuge will cause a vacuum to form in your sample; when removing the parafilm, this can cause the membraneto be lifted out of the o-ring that holds it in place, causing your sample to spill out the bottom!

3. Remove the NMR sample from the centricon (by inverting it and by centrifuging). Transfer to a 5 mm Shigemi NMR tube. Rinse the centricon with NMR buffer (once or twice) so that the final volume of the NMR sample is 320 uL.

4. Seal the NMR tube first with teflon tape and then with parafilm.


In order to facilitate the preparation of NMR samples, UTHSCSA general stores now carries a)99.9% D2O and b) 5 mm Shigemi tubes for Bruker probes (as of July, 2003). The D2O is sold in 100 mL bottles and the order ID is 175013788. The Shigemi tubes are sold in a quantity of one and the order ID is 175042166.If you need larger quantities, you might contact one of the stable isotope suppliers or Shigemi directly.


Previous Page Next Page

People Searching On This Page:
  • phosphate buffer for nmr
  • nah2po4 buffer nmr
  • stability guid

Related Pages


Preparation of Implants

Preparation of Implants

Articles | Pharmaceutics
Date:
04-Jul-2011  Views: 6460

Although many polymers can by used to prepare rate-limiting membranes for controlled release relatively few are employed for implantation purpose beca ...
Preparation of NanoSuspensions

Preparation of NanoSuspensions

Articles | Pharmaceutics | Suspensions
Date:
02-Jun-2011  Views: 4654

Nanosuspensions can be produced by bottom-up or top-down techniques. In thecase of bottom-up technique, the strategy is to build particles from their ...
Basics of NMR Sprectroscopy

Basics of NMR Sprectroscopy

Articles | Analysis | Sprectroscopy
Date:
24-Apr-2011  Views: 3952

Nuclear magnetic resonance spectroscopy, commonly referred to as NMR, has become the preeminent technique for determining the structure of organic com ...
Theory  of NMR Spectroscopy

Theory of NMR Spectroscopy

Articles | Analysis | Sprectroscopy
Date:
24-Apr-2011  Views: 3762

The phenomenon of magnetic resonance results from the interaction of the magnetic moment of an atomic nucleus (µ) with an external magnetic field. The ...
Preparation of Effervescent tablets

Preparation of Effervescent tablets

Articles | Pharmaceutics | Tablet
Date:
29-Mar-2011  Views: 37416

RAW MATERIALSThe effervescent tablets, when added to water, generate a gas, which causes effervescence and produces a clear and fresh tasting, sparkli ...
Post Your Comments (No Login Require)
Name : (required)
Email : (required)
Website :

Comment : (required)

76  + 8 =     
Comments
People Searched About:
Phosphate Buffer For Nmr   |   Nah2po4 Buffer Nmr   |   Stability Guid   |  
Google : 85 times | Yahoo : 10 times | Bing : 215 times |