PROTOCOLS
High Resolution Sample Preparation Guide
Strong acids contain metal ions and organic solvents. Dry the tubes by blowing them with dry N2 and put them in a clean oven at < 50 oC. Note: Exceeding 50 oC will ruin the tube.
For non-aqueous solvents one should use 100% deuterated solvent (e.g., DMSO-d6) to avoid a large solvent background signal. Keep the tube and the solvent dry, avoid exposure to the air. If your sample is dilute (<5 mM) it is a good idea to prepare a second sample tube containing just the solvent so that you can subtract the signals arising from solvent impurities.
For aqueous solutions one should use 100% D2O whenever possible. However, you will usually have to use undeuterated H20 in order to observe labile protons (e.g., amide protons in proteins). In this case use 90% H2O / 10% D20, so that there is a sufficient deuterium signal for the lock system to function.
Buffer solutions should not contain paramagnetic ions and should have some total salt concentration < 200 mM. Any organic component of the buffer solution (e,g., Tits, acetate, etc.) in. excess of 500 uM should be deuterated to avoid background signals.
For work where accurate measurement of chemical shifts is needed an internal standard should be used. In organic solution’s one can use tetramethylsilane (TMS). In aqueous solutions one can use 3,3,3-trirnethylsilylprupionate (TSP), or 5,5-dimethylsilapentanesulfonate (DSS).
Bacterial growth can degrade your sample over time. If you have sample degradation, use > 80% D20, or use a cheater (e.g., EDT A, EGA) to complex divalent cations, or add NaN3 (< 50 um) to your buffer, or use micro porous filtration as the last step in your sample preparation.
Paramagnetic ions (e.g., Fe3.+, Mn2+) can cause large distortions in chemical shifts and relaxation times if they form complexes with your molecule. Solvated paramagnetic ions will also cause broadening of the water signal, which will severely compromise solvent suppression. Buffer solutions must not contain these ions, however, they are often found in small concentrations as impurities. One should avoid chromic acid or other metal-containing oxidants for tube cleaning. To sequester paramagnetic ions add 50 uM EDTA and/or remove paramagnetics by incubating with Chelex.
Animal Anesthesia Guidelines
Ensure that there is adequate liquid anesthetic (ISOFLURANE) in the vaporizer.
Open the oxygen ( O2 ) cylinders completely and ensure that you have adequate O2 to complete your experiment. Each cylinder holds approximately 6– liters of O2 when full (200 PSI). The amount of O2 left in the cylinder is proportional to the remaining gas pressure.
Check the active scavenging system and turn the suction pump on if the active scavenge system is necessary. Anesthetic gas can be passively scavenged if the activated charcoal canister is removed from the rubber sleeve and placed outside the suction pump apparatus. Each charcoal canister can be used for 12 hours before replacement is necessary. Induce patients using higher fresh gas flow rates (O2 flow as indicated by the O2 flow meter ). Flow rates should be approximately three to four liters per minute during induction in chambers or via face masks. Isoflurane vaporizer settings should be at 4% to 5 % for induction only. Turn off the O2 flow when opening the chambers or removing face masks. Patients may arise quickly when removed from chambers or face masks. Therefore, have everything ready before you induce your animal patient.
Once patients are induced to anesthesia, placed into the probe and the anesthetic breathing hoses are attached properly to the probe, the isoflurane vaporizer setting can be reduced to 1% to 1.5% and the O2 flow rate can be reduced to 1.5 liters per minute.
When turning off the anesthetic, turn the vaporizer dial all the way past 0 (zero) until it “clicks” off. Wait approximately one to two minutes, then turn the O2 flow meter off. The needle valve to the O2 flow meter is delicate and should not be forced completely shut. Stop turning it when the float (ball) falls to the bottom.
Turn off the O2 E cylinder and flush the pressure from the system using the O2 flush valve. This takes approximately 10 seconds.
