1D NMR Titrations

Protein Stock Preparation

1. Make 2.0 mM (2000 uM) HSA stock solution
   1. Dissolve 130.8 mg of HSA (MW = 65400 g) in 1.0 mL D₂O
2. 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)
3. Spin Samples down in mini centrifuge
4. Press short button until max speed then release
5. Measure the absorbance and generate a standard curve for the new samples.
6. Make 10X dilutions (10.0 uL HSA and 990 uL D₂O)

Table 1:

Sample No.	NMR [HSA] (uM)	[HSA] (uM)	Volume (n+1) sample (uL)	Volume of D2O (uL)
1	0.000	0.00	0	0
2	0.100	5.00	500	500
3	0.200	10.00	500	500
4	0.400	20.00	667	333
5	0.600	30.00	750	250
6	0.800	40.00	800	200
7	1.000	50.00	500	500
8	2.000	100.00	667	333
9	3.000	150.00	750	250
10	4.000	200.00	667	333
11	6.000	300.00	750	250
12	8.000	400.00	800	200
13	10.00	500.00	833	167
14	12.00	600.00	857	143
15	14.00	700.00	875	125
16	16.00	800.00	889	111
17	18.00	900.00	900	100
18	20.00	1000.00	667	333
19	30.00	1500.00	750	250
20	40.00	2000.00	130.8 mg	1 mL

Ligand Stock Preparation

1. Make 50.0 mM Potassium Phosphate buffer at pH 7.0 in D₂O
   1. 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.
   2. Spike with 500 uL of TMSP from 10.0 uM stock solution
2. Make 500.0 uL fresh ligand stocks at 20.0 mM concentration
   1. Calculate the mass of ligand needed and dissolve in 500.0 uL of 100% DMSO
3. Make 11.0 mL titration stocks
   1. 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.
   2. 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)
4. Vortex the titration stock solutions to mix

NMR Sample Preparation

1. Label 20 1.5 uL centrifuge tubes with the ligand and protein concentration
2. To each tube add 490 uL of the ligand titration stock solution
3. 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)
4. Spin samples down in mini-centrifuge to make sure all sample is in the bottom of tube
5. Pipette sample out of centrifuge tubes and into NMR tubes making sure not to get any bubbles
6. Run samples on NMR using the zgesgp pulse program (1D-FASTNMR parameter set) or other comparable method for water pre-saturation

Important Notes

• 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