Protein Melting Temperature (Tm) Analysis

Protein Melting Temperature (T_m) is a critical biophysical parameter that quantifies the thermal stability of a protein. It represents the temperature at which 50% of the protein undergoes irreversible unfolding, transitioning from a folded, functional conformation to a denatured state. In biopharmaceutical development, T_m serves as a key indicator of structural integrity, formulation robustness, and product shelf-life. Proteins with higher T_m values generally exhibit greater resistance to thermal stress, aggregation, and degradation - factors essential for ensuring the efficacy and safety of biologic drugs.

Protein melting temperature and its measurement applications diagram. (STEMart Original)

Our Methods

Our workflow for protein melting temperature analysis. (STEMart Original)

Differential Scanning Calorimetry (DSC)

DSC measures the heat absorbed or released during protein unfolding as a function of temperature. The T_m corresponds to the peak of the heat capacity curve.

It directly quantifies thermal transitions without the need for labels or probes. Ideal for high-purity proteins, DSC provides thermodynamic parameters (ΔH, ΔS) alongside T_m. It requires larger sample volumes (~0.5–1 mg/mL) compared to other methods.

Circular Dichroism (CD) Spectroscopy

CD detects changes in secondary structure (α-helix, β-sheet) by measuring the differential absorption of left- and right-circularly polarized light. Thermal denaturation curves are generated by monitoring signal loss at structural-sensitive wavelengths.

This method combines T_m determination with structural insights. It is suitable for low-concentration samples but is limited to optically transparent buffers.

Dynamic Light Scattering (DLS)

DLS tracks changes in hydrodynamic radius (Rh) due to protein unfolding or aggregation. A sharp increase in Rh at elevated temperatures correlates with T_m.

It provides rapid screening for aggregation-prone proteins. DLS requires minimal sample preparation but may lack resolution for subtle stability differences.

Fluorescence Thermal Shift Assay (FTSA)

FTSA uses environment-sensitive fluorescent dyes that bind to hydrophobic regions exposed during unfolding. Fluorescence intensity increases with temperature, and T_m is derived from the inflection point of the sigmoidal curve.

This method offers high-throughput, low-sample-volume analysis and is compatible with 96/384-well plates. It is widely used for screening buffer conditions or ligand effects.

Industry Standards

ICH Q6B: Recommends T_m analysis for confirming higher-order structure of biotherapeutics.
USP <1049>: Provides guidelines for calorimetric methods in quality control.

By integrating T_m analysis into biopharmaceutical workflows, STEMart provides developers actionable insights to design stable, manufacturable, and compliant protein therapeutics. For more information on our T_m analysis service or to discuss your specific requirements, please contact us today.