1D NMR Titrations
Protein Stock Preparation
- Make 2.0 mM (2000 uM) HSA stock solution
- Dissolve 130.8 mg of HSA (MW = 65400 g) in 1.0 mL D2O
- Make a set of serial dilutions starting with the 2.0 mM stock (see table 1) (NOTE: at these stock solutions it only takes 10.0 uL of protein sample to give the desired protein concentration in the NMR tube)
- Spin Samples down in mini centrifuge
- Press short button until max speed then release
- Measure the absorbance and generate a standard curve for the new samples.
- Make 10X dilutions (10.0 uL HSA and 990 uL D2O)
|Sample No.||NMR [HSA] (uM)||[HSA] (uM)||Volume (n+1) sample (uL)||Volume of D2O (uL)|
|20||40.00||2000.00||130.8 mg||1 mL|
Ligand Stock Preparation
- Make 50.0 mM Potassium Phosphate buffer at pH 7.0 in D2O
- For 500.0 mL total solution, measure 2.177 g K2HPO4 (MW = 174.18 g/mol) and 1.70 g KH2PO4 (MW = 136.09g/mol) in separate 250.0 mL volumetric flasks and fill to line with 99.9% D2O.
- Spike with 500 uL of TMSP from 10.0 uM stock solution
- Make 500.0 uL fresh ligand stocks at 20.0 mM concentration
- Calculate the mass of ligand needed and dissolve in 500.0 uL of 100% DMSO
- Make 11.0 mL titration stocks
- Single ligands: In Falcon tube add, in order, 10.5 mL Potassium Phosphate buffer, 440 uL 100% DMSO and finally 55 uL of fresh ligand stock.
- Ligand Mixtures: In a Falcon tube add in order: 10.390 mL Potassium Phosphate buffer, 440 uL 100% DMSO and finally 55μL of each fresh ligand stock solution (The 2 non-binders Choline Bromide and Uridine-5’-monophosphate plus the binding ligand)
- Vortex the titration stock solutions to mix
NMR Sample Preparation
- Label 20 1.5 uL centrifuge tubes with the ligand and protein concentration
- To each tube add 490 uL of the ligand titration stock solution
- To each tube add 10 uL of the specific protein stock to the intended sample (i.e. add 10 uL of 400 uM stock to get protein concentration in NMR tube of 8 uM)
- Spin samples down in mini-centrifuge to make sure all sample is in the bottom of tube
- Pipette sample out of centrifuge tubes and into NMR tubes making sure not to get any bubbles
- Run samples on NMR using the zgesgp pulse program (1D-FASTNMR parameter set) or other comparable method for water pre-saturation
- This is a general method for preparing samples for NMR titrations and can be amended as needed.
- It has been found that the best protein concentration range is 0.00 uM to 40.0 uM to get a complete binding curve for the majority of ligands. This is only for HSA and is most likely different for different proteins.
- Solution Human Serum Albumin is only good for about fourteen days, make sure to write down the date made and make new sample every 14days as needed