Key Points for Measuring Proteins and Biopolymers with DSC (Differential Scanning Calorimetry)

MicroCal PEAQ-DSC (Differential Scanning Calorimeter) for Biomolecular Stability Evaluation is used to convey key points when measuring proteins and biopolymers with DSC (Differential Scanning Calorimetry).

What should you be mindful of to obtain high-quality DSC data? What is the state of the sample? What are the measurement conditions? Let’s reconfirm with the following list!

[Sample Conditions]

To acquire high-quality DSC data, please adhere to the following items.

• Match the composition of the buffer in the sample cell and reference cell.
• Adjust samples to the desired buffer using dialysis, desalting columns, or other buffer exchange methods.
• Keep the dialysate and buffer used during the buffer exchange for use in the reference cell and buffer-buffer scans (hereafter referred to as buffer scans).
• The recommended protein sample concentration is 0.1–2 mg/mL.
• The T_m (midpoint of denaturation) obtained from DSC is concentration-dependent. Therefore, when performing T_m screening or equivalence evaluation, match all sample concentrations.

[Measurement Condition Settings]

• When performing T_m screening or equivalence evaluation with DSC, all measurement conditions (scan rate, temperature range, feedback mode, pre-scan thermostat, etc.) must be consistent. The shape of the thermogram is influenced by the scan rate and feedback mode.
• Temperature range: Set the starting temperature to be 10–20°C lower than the first T_m value and the end temperature to be 10–20°C higher than the last T_m value.
• Scan Rate (general recommendations)
  • Proteins, nucleic acids: 60–90°C/h
  • Lipids: 30–90°C/h
  If you want to increase throughput for T_m screening, measure at 180–240°C/h (compatible models: PEAQ-DSC, VP-Capillary DSC).
• Feedback Mode
  • Proteins: none or low
  • Nucleic acids: low or medium
  • Lipids: high
• Pre-scan thermostat: Suggested duration is 3–15 minutes.

[Notes on Systems with Autosamplers]

• Consider the stability of the sample and store it at an appropriate temperature. If the sample is a protein or other biological macromolecule, set the cooling stack temperature to 5–10°C. If the sample is stable, 25°C is acceptable.
• Set Purge/Refills (or the number of filling strokes) to at least 3 times.
• Incorporate detergent washing with 14% Decon90 (or 20% Contrad70) into the method.

[Quality of Raw Measurement Data]

• Always perform sample scans and buffer scans under the same scan conditions. If necessary, a buffer scan can be carried out before each sample.
• Frequent buffer scans help evaluate whether the cell is clean and the instrument is functioning well.
• If you always organize the measurement schedule under the same scan conditions, the reproducibility of buffer scans should be high.
  (The PEAQ-DSC Automated is equipped with an efficient wash pump and optimized wash protocols. It enables the continuous measurement of 3 or more samples without inserting buffer scans between sample measurements.)
• The DP value (mcal/min) at the start of buffer scans and water-water scan measurements should be as close to zero as possible.
• During protein sample measurements, the DP value becomes negative compared to buffer scans. Higher concentrations of protein result in larger negative values. When there is minimal deviation in buffer composition, the deviation between sample scans and buffer scans is approximately 0.15–0.25 mcal/min for a protein concentration of 1 g/L. (Figure 1)

If there is a deviation of 1 mcal/min or more in the DP value between the sample scan and buffer scan, the following reasons may be considered. (Figure 2)

• Mismatch occurred between the sample and buffer
• The cell was dirty
• Insufficient filling of the solution
• Air bubble inclusion
• Very high concentration sample