Sample Preparation for 4D NMR Experiments

This tutorial outlines the procedure for preparing protein samples for 4D NMR experiments, assuming the starting material is glycerol-free. If your protein aliquots contain glycerol, please follow the separate glycerol removal tutorial.

The process assumes you are starting with a series of protein aliquots (e.g., 6 × 100 μL, 300 μM assumed) and guides you through:

• Concentration of the protein solution
• Measurement of protein concentration
• Addition of Acetyl-CoA (AcCoA) to achieve a 1:1 molar ratio (if applicable)
• Final transfer into an NMR tube (Shigemi type)

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Equipment and Materials

• Fixed-angle centrifuge rotor
• Pipettes (ideally in 100 μL and 200 μL ranges)
• 0.5 mL centrifugal filter unit (3–100 kDa MWCO), placed inside a support Eppendorf tube
• NMR buffer (glycerol-free), e.g.: 25 mM Tris, 300 mM NaCl, 1 mM TCEP, pH 7.5
• Protein aliquots (e.g., 6 × 100 μL, 300 μM assumed)
• Ice box
• NanoDrop spectrophotometer (for concentration measurement — see separate tutorial)
• D₂O solution
• Shigemi tubes

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1. Concentration of the Protein Solution

The goal of this step is to reduce the combined volume of your aliquots (~600 μL) to approximately 350 μL, which is enough to fill a Shigemi NMR tube (~300 μL) while providing some extra volume for measurement and transfer loss.

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🔹 Step 1: Pre-wash the Filter Unit

Since the filter unit lacks volume markings, you will need to add one manually:

1. Pipette 350 μL of NMR buffer into the filter unit.
2. Use a marker to draw a line at the 350 μL level. This mark will serve as a reference for future volume adjustments.
3. Pipette additional 150 μL into the filter unit to reach its full capacity.
4. Insert the unit into the centrifuge rotor.
5. Centrifuge at 8,000 × g for 10 minutes at 4 °C.
6. If the buffer still remains in the filter unit, centrifuge again for 5–10 minutes until the unit is empty.

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🔹 Step 2: Pool the Protein Aliquots

1. Remove 6 protein aliquots from the freezer and thaw them on ice for approximately 10–15 minutes.
2. Once thawed, pipette the contents of the first five aliquots directly into the sixth one.
3. Vortex the tube briefly to ensure thorough mixing.
4. You should now have approximately 600 μL of sample, accounting for minor pipetting losses.

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🔹 Step 3: Concentrating the Sample

In this step, you will use the 350 μL mark made earlier to estimate the volume during concentrating.

1. Pipette 500 μL of your protein sample into pre-washed filter unit.
2. Centrifuge the sample at 8,000 × g for 1 minute at 4 °C.
3. After each spin, check if the volume in the filter unit is above mark.
   • If the volume is still above the target, continue centrifugation in 1-minute increments.
   • Stop once the volume matches the reference level.

⚠️ Avoid overdrying the sample — stop slightly above the target volume if in doubt.

💡 If your total sample volume exceeds the filter unit’s capacity (e.g., ~600 μL total), process the remaining volume like this:

• After reducing the first 500 μL to ~400–450 μL, add the remaining ~100 μL directly into the same filter unit.
• Continue centrifugation in 1-minute increments as before.
• This ensures that all of your protein sample is retained and concentrated together without needing a second filter unit.

1. Place the filter unit with the concentrated solution with the NMR buffer on ice and head to the NanoDrop station to measure concentration.

2. Concetration Measurement

Measure the concetration of your sample by following the NanoDrop tutorial. This concetration will serve as a starting point for the calculation of how much AcCoa is needed to be added to achieve the desired ratio between protein and cofactor (if needed).

3. Final NMR Sample preparation

After the concetration measurement, transfer 300 μL of the concentrated sample into the Eppendorf tube. In this way, you will establish the volume of your protein stock for calculation of how much AcCoa and D₂O to add. If you need to add AcCoa, folow from Step 1, otherwise jump to Step 2

🔹 Step 1: Calculation of the added volume

To determine how much AcCoA stock to add to your sample to reach a 1:1 molar ratio (AcCoA:protein), use the formula below.

V_{AcCoA}~[μL] = \frac{C_{protein}\times V_{protein}\times 10^{-3}}{C_{AcCoA}}

Where:

• V_AcCoA = volume of AcCoA to add (in μL)
• C_protein = protein concentration (in μM)
• V_protein = protein sample volume (in μL)
• C_AcCoA = AcCoA stock concentration (in mM)

⚠️ Units required for this formula:

• Protein concentration in μM
• Protein volume in μL
• AcCoA stock concentration in mM
• Resulting AcCoA volume in μL

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💡 Example Calculation

Assume you have:

• C_protein = 400 μM
• V_protein = 300 μL
• C_AcCoA = 8 mM

Plug the values into the formula:

V_{AcCoA} = \frac{400 × 300 × 10^{-3}}{8} = \frac{120}{8} = 15 μL

✅ Result: Add 15 μL of 8 mM AcCoA to your protein sample to reach a 1:1 molar ratio.

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🔹 Step 2: Addition of D₂O

To enable frequency locking during NMR acquisition, add D₂O to the sample to reach a final content of 5–10%. A common choice is 7%.

Use the following formula to calculate the volume of D₂O to add:

V_{D2O}~[μL] = \frac{p \times V_0}{1 - p}

Where:

• V_D2O = volume of D₂O to add (in μL)
• V₀ = current sample volume before adding D₂O (in μL)
• p = desired D₂O percentage as a decimal (e.g. 0.07 for 7%)

💡 Example Calculation

Let’s say:

• Current volume = 315 μL (300 μL protein + 15 μL AcCoA)
• Desired D₂O fraction = 7% (i.e. p = 0.07)

Plug the values into the formula:

V_{D2O}~[μL] = \frac{0.07 \times 315}{1 - 0.07} \approx 23.7 μL

✅ Result: Add 23–24 μL of D₂O to your sample to reach ~7% D₂O content.

This gives you a final volume of ~339 μL, suitable for Shigemi tube preparation.

🔹 Step 3: Final Steps

• Vortex the tube briefly to ensure thorough mixing.
• Transfer the sample into the Shigemi tube using the glass Pasteur pipette.
• Cap the Shigemi tube.
• Spin the Shigemi tube on the table rotor to get rid of the bubbles.

The following steps should be performed by NMR facility personnel but are included here for completeness:

• Insert the plunger so the meniscus is 2–3 mm below the rim. Be careful, no bubble must be present in the tube.
• Cap, clean the exterior, and load the tube into the spectrometer.