Binding Affinity (Kd) Measurement
Accurately quantify how tightly two molecules bind—essential for lead validation, SAR studies, and hit optimization.
Isothermal Titration Calorimetry (ITC) Analysis Service
Quantify Molecular Interactions with High Sensitivity and Unmatched Thermodynamic Precision
Creative Proteomics offers label-free, in-solution isothermal titration calorimetry (ITC) services to quantify binding affinity, stoichiometry, and thermodynamics (Kd, ΔH, ΔS, ΔG) in one assay. Ideal for drug discovery, protein engineering, and molecular interaction studies.
Why Choose Us:
- Accurate affinity measurement from nM to mM
- Full thermodynamic profiling in native conditions
- Low sample volume, high sensitivity
- Expert-designed experiments and clear interpretation
Get trusted data. Make confident decisions.
What Is Isothermal Titration Calorimetry?
Isothermal Titration Calorimetry (ITC) is a powerful, label-free binding assay that directly measures the heat released or absorbed during molecular interactions—allowing researchers to characterize binding affinity (Kd), stoichiometry (n), enthalpy (ΔH), and entropy (ΔS) in a single experiment.
Unlike techniques that require labeling or immobilization, ITC analyzes interactions in native solution conditions, preserving molecular conformation and providing a complete thermodynamic profile. This makes ITC ideal for studying a wide range of biomolecular systems, including protein–ligand, protein–protein, RNA–small molecule, and peptide–receptor interactions.
ITC offers unique insight into the driving forces behind binding—whether it's enthalpy-driven, entropy-driven, or a combination of both.
Typical Scientific Questions ITC Can Answer
- Is my lead compound binding tightly enough to the target protein?
- Does a mutation affect binding strength or thermodynamic balance?
- Is the interaction enthalpy- or entropy-driven?
- What is the ideal binding stoichiometry for my system?
- Can I compare thermodynamic signatures between candidates?
If your project depends on precise, mechanistic insight into binding interactions, ITC is the gold standard.
Why Choose Our ITC Service?
Sensitive Detection Across a Broad Affinity Range
Our MicroCal iTC200 platform enables accurate measurement of binding affinities ranging from nanomolar (nM) to millimolar (mM), ideal for weak and strong interactions alike.
- Detect Kd values as low as 10 nM, up to 10 mM—no label required.
High Precision with Minimal Sample Volume
We work with limited and valuable samples without compromising data quality.
- Requires as little as 300 μL per sample, with protein concentrations as low as 5–10 μM.
- Data variation typically <1% RSD across replicates.
Full Thermodynamic Profiling in One Experiment
Each assay outputs a complete set of binding parameters, eliminating the need for multiple techniques:
| Parameter Measured | Value Range (Typical) |
| Kd | 10-9 to 10-3 M |
| Stoichiometry (n) | Single or multiple sites |
| ΔH, ΔS, ΔG | Reported in kcal/mol |
Strict Buffer Matching and Quality Control
To minimize background noise, we provide buffer matching, degassing, and filtration services, ensuring thermal baseline stability within ±0.1 μcal/sec.
Experienced Biophysics Team
Our scientists have completed over 300 successful ITC projects, covering protein–ligand, protein–peptide, and RNA–small molecule systems across multiple industries.
Technical Services
What We Offer
Workflow and Instrumentation
Sample Requirement
ITC vs Other Binding Assays
Deliverables
Case Study
FAQ
Get a Custom Proposal
What ITC Analysis Services Do We Offer?
At Creative Proteomics, we offer a comprehensive suite of isothermal titration calorimetry (ITC) analysis services to help you understand biomolecular interactions from multiple scientific angles. Whether you're investigating a small molecule inhibitor, optimizing antibody affinity, or characterizing RNA–protein interactions, our ITC platform provides precise thermodynamic and kinetic insights.
Here’s what you can analyze with our ITC service—and the specific research problems we help you solve:
Binding Stoichiometry (n)
Determine the molar binding ratio to reveal interaction models (1:1, 1:n, cooperative binding, etc.).
Thermodynamic Profiling (ΔH, ΔS, ΔG)
Understand the driving forces behind binding—discriminate between enthalpy- or entropy-driven interactions.
Mutant vs. Wild-Type Binding Comparison
Evaluate how mutations affect binding behavior and thermodynamic balance—crucial for protein engineering.
Small Molecule–Protein Interaction Analysis
Assess direct binding of drug candidates to protein targets without the need for labels or immobilization.
RNA/DNA–Ligand Binding Studies
Characterize nucleic acid interactions with small molecules, peptides, or proteins under native conditions.
Mechanism-of-Action (MoA) Support
Reveal binding mechanisms, conformational changes, and cooperative effects to support functional hypotheses.
Every ITC project is fully customizable. Our scientists can help you design the optimal experiment based on your sample type, expected binding range, and research goals.
Our ITC Workflow
1
Sample Quality Check & Preparation
Ensure sample purity, concentration accuracy, and buffer compatibility. Proper buffer matching minimizes baseline noise and heat artifacts.
2
Instrument Setup & Titration Execution
Titration is performed using high-precision calorimeters (e.g., MicroCal iTC200). The ligand is injected stepwise into the protein solution under constant temperature and stirring.
3
Data Acquisition
Each injection produces a measurable heat change. Real-time thermograms are integrated to generate binding isotherms.
4
Thermodynamic Modeling
Data are fitted to binding models (1:1, multiple sites, etc.) to determine Kd, ΔH, ΔS, and stoichiometry (n). This provides a full thermodynamic profile.
5
Report Delivery
Clients receive raw and processed data, fitted curves, and a complete thermodynamic table with expert interpretation—ideal for drug discovery, binding affinity ranking, and mechanistic studies.
ITC Instrumentation & Technical Capabilities
MicroCal iTC200
– Precise thermal sensitivity (as low as 0.1 μcal/sec)
– 200 μL sample cell capacity
– Fully automated injection system (40–60 μL syringe)
– Temperature control range: 2–80°C
Nano ITC Standard Volume
– Designed for ultra-low-volume samples with high sensitivity
– Compatible with a wide range of molecular weights and binding affinities
These instruments enable accurate thermodynamic profiling for a broad range of interaction strengths (nM to mM Kd).
Sample Requirements for Isothermal Titration Calorimetry Analysis
| Parameter | Recommended Specifications |
| Sample Types | Proteins, peptides, small molecules, RNA, DNA |
| Purity | ≥ 95%; free from aggregates, particulates, and enzymatic degradation |
| Concentration (Cell) | 5–50 μM (typically); optimized depending on Kd and molecular weight |
| Concentration (Syringe) | 10× to 20× higher than the cell sample (e.g., 100–500 μM) |
| Volume Required | ≥ 300 μL for each sample (cell and syringe); additional volume for replicates recommended |
| Buffer Conditions | Tris, HEPES, PBS, phosphate buffer, or custom buffers (avoid exothermic buffer mismatches) |
| Buffer Matching | Both titrant and titratee must be in identical buffer systems to avoid heat of dilution |
| Additives | DMSO ≤ 2% tolerated; detergents must be assessed for compatibility |
| Filtration | 0.22 μm filtration recommended to remove particulates |
| Degassing | All samples should be degassed prior to analysis to avoid air bubbles |
Not sure your sample is ready? We offer buffer exchange, concentration adjustment, and quality check services upon request.
ITC vs Other Binding Assays: Which One Fits Your Study?
| Feature / Method | ITC (Isothermal Titration Calorimetry) | SPR (Surface Plasmon Resonance) | BLI (Bio-Layer Interferometry) | MST (Microscale Thermophoresis) |
| Label-Free | ||||
| In-Solution Measurement | ||||
| Binding Affinity Range | ~10⁻⁹ to 10⁻³ M | ~10⁻¹² to 10⁻⁶ M | ~10⁻¹² to 10⁻⁶ M | ~10⁻¹² to 10⁻³ M |
| Thermodynamic Data (ΔH, ΔS, ΔG) | ||||
| Kinetic Data (ka, kd) | ||||
| Multivalent/Allosteric Binding | Limited depending on setup | Limited | ||
| Sample Volume Requirement | Moderate (~300 μL) | Low (~50–100 μL) | Low (~50–100 μL) | Very Low (~5–10 μL) |
| Buffer Compatibility | Strict (matching required) | Flexible | Flexible | ⚠️ Sensitive to additives |
| Immobilization Required | ||||
| Sample Labeling | ||||
| High-Throughput Capable | Medium (automated SPR available) | |||
| Data Interpretation Complexity | Medium to high (thermodynamic modeling) | Medium (ka/kd curves) | Medium | Low to medium |
| Best Used For | Mechanism studies, thermodynamics | Kinetics, concentration screening | Epitope binning, affinity ranking | Interaction screening, rapid affinity analysis |
Summary Recommendations
- Choose ITC if your priority is to understand why molecules bind—i.e., thermodynamic profiling (ΔH, ΔS, ΔG) and stoichiometry in native solution.
- Choose SPR if you need real-time kinetics (ka/kd) or are comparing large sets of compounds with moderate sample volume.
- Choose BLI for higher throughput screening with relatively lower setup cost and surface sensitivity than SPR.
- Choose MST for low-volume, low-affinity systems, especially early-stage fragment hits or precious samples requiring fluorescent tagging.
Applications of ITC Analysis
Versatile Binding Analysis for Drug Discovery, Structural Biology, and Beyond
Drug Discovery
Validate small molecule–target binding, rank compound affinities, support hit-to-lead optimization
Protein Engineering
Compare binding thermodynamics of wild-type vs. mutant variants
Signal Transduction Research
Characterize interactions between signaling proteins or receptors and their regulatory partners.
Synthetic & Systems Biology
Validate designed protein–protein or protein–RNA modules by profiling their binding thermodynamics.
Plant Molecular Biology
Study interactions involved in developmental signaling, environmental responses, or plant–pathogen systems.
Macromolecular Assembly
Analyze assembly energetics in multicomponent protein or nucleic acid complexes under near-native conditions.
Deliverables: What You’ll Receive
Clear, Interpretable, and Publication-Ready Binding Data
When you partner with Creative Proteomics for ITC analysis, you receive more than just raw output—we provide a fully interpreted, publication-ready results package that supports both scientific insight and downstream use.
Raw Thermogram Data
Real-time power vs. time curves recorded for each injection cycle, ideal for baseline inspection and traceability.
Integrated Heat Plot
Molar ratio vs. injection heat curve, used for isotherm fitting and stoichiometry analysis.
Fitted Binding Isotherms
Overlay of fitted binding curves with calculated Kd, ΔH, ΔS, ΔG, and n values. Includes curve fit confidence and residuals.
Complete Thermodynamic Summary
Numerical output table with fitted values, errors, and model statistics—ready for publication or filing.
Experimental Conditions Report
Includes sample concentrations, syringe setup, injection volume, temperature, buffer components, and pH
Expert Interpretation Notes
Insightful annotations on data quality, model selection, binding mechanism, and next-step recommendations.
Case Study
Case 1
Case 2
Case 1: NRG1C vs NRG1A Binding to EDS1–SAG101 Complex
Research Objective:
To investigate how NRG1C dynamically regulates plant immunity by competing with NRG1A for binding to the EDS1–SAG101 complex.
How ITC Was Used:
- Method: ITC (MicroCal PEAQ-ITC) was used to measure binding affinity between EDS1–SAG101 and NRG1C or NRG1AΔCC.
- Conditions: Tris-HCl buffer, 25°C, injections of ligand into sample cell containing receptor.
Key Findings from ITC:
- NRG1C binding Kd ≈ 43 nM, much tighter than NRG1AΔCC Kd ≈ 4 μM.
- This demonstrates that NRG1C has ~100x higher affinity, supporting a competitive inhibition model for immune regulation.
Why ITC Was Essential:
- Quantified differences in binding strength.
- Supported mechanistic insights into competitive regulation of immune responses at molecular level.
Additional Techniques:
GST pull-down validated competition, confirming ITC findings.
Reference
Huang, Shijia, et al. "Balanced plant helper NLR activation by a modified host protein complex." Nature (2025): 1-9. https://doi.org/10.1038/s41586-024-08521-7
a. Structural model showing bulky residues of NRG1C interacting with the EP domain of SAG101.
b. Co-expression assays using SAG101 mutants (EFI/REE and HER/REE) with EDS1, NRG1C, RPP1, and ATR1.
c. ITC quantification of ADRr-ATP–bound EDS1–SAG101 binding to NRG1C and NRG1AΔCC.
d. Competition assay showing NRG1C disrupts EDS1–SAG101 binding to NRG1AΔCC in a dose-dependent manner.
Case 2: Binding of RALF23 to FER and LLG1 in Receptor Complex Formation
Research Objective:
To elucidate how the RALF23 peptide induces assembly of a receptor complex involving FER and LLG1 proteins in plants.
How ITC Was Used:
- Method: ITC200 (MicroCal LLC) used to test direct and indirect interactions among RALF23, FER^ECD, and LLG1.
- Conditions: Bis-Tris buffer, pH 6.0, 25°C.
Key Findings from ITC:
- FER^ECD–RALF23 Kd ≈ 1.52 μM
- LLG1–RALF23 Kd ≈ 4.95 μM
- No binding detected between FER^ECD and LLG1 alone.
- Mutational analysis (G123R) showed reduced affinity (Kd ≈ 21.4 μM), confirming a key binding site.
Why ITC Was Essential:
- Clarified interaction specificity and strength.
- Validated mutational effects on ligand recognition.
- Provided biophysical evidence for ligand-induced receptor complex formation.
Additional Techniques:
Structural biology (X-ray), immunoprecipitation complemented and validated ITC results.
Reference
Xiao, Yu, et al. "Mechanisms of RALF peptide perception by a heterotypic receptor complex." Nature 572.7768 (2019): 270-274. https://doi.org/10.1038/s41586-019-1409-7
Quantification of binding affinities by ITC assays. Left, LLG1 was titrated into FERECD. Middle and right, RALF23 peptide was titrated into FERECD or LLG1. The binding constants (Kd values ± fitting errors) and stoichiometries (n) are indicated. ND, no detectable binding.
You May Want to Know
What if I don’t know the expected binding affinity (Kd) of my system?
No problem. Our scientists will help estimate the appropriate concentration ranges and injection settings based on your molecule types and interaction hypotheses. We also offer pre-study consultation to help you avoid under- or over-saturation conditions.
Can ITC be used for very tight or very weak binding interactions?
ITC is most accurate within a Kd range of ~10 nM to 10 mM. For very tight binders (<1 nM), competitive binding or reverse titration methods may be applied. For very weak interactions, we may recommend combining ITC with orthogonal techniques such as SPR or BLI.
Do I need to match buffer components between the two interacting molecules?
Yes—buffer matching is essential. Even minor differences in salt or pH can produce significant heats of dilution that obscure binding signals. We offer buffer exchange and dialysis services to help align buffer conditions between samples.
Can ITC detect non-specific interactions?
Yes, and this is often a valuable feature. Non-specific binding may show up as irregular heat profiles, poor curve fitting, or abnormal stoichiometry. Our analysis includes data interpretation to flag such effects and, when appropriate, suggest alternative assay designs.
How do I know if my molecule is stable enough for ITC?
ITC is conducted at constant temperature with stirring, so molecular integrity is key. We recommend running basic stability tests such as DLS or SDS-PAGE before submission. If needed, we can evaluate aggregation risk during sample preparation.
Is it possible to analyze multivalent or allosteric binding using ITC?
Yes. ITC can support multivalent, cooperative, and sequential binding models. In such cases, curve fitting may require more complex models, and our team will advise accordingly.
What if I only have a limited amount of sample?
We can work with as little as 300 μL per sample, but lower volumes may restrict titration cycles or reduce reproducibility. Let us know your constraints and we’ll suggest a minimized injection plan or concentration optimization strategy.
Can I use DMSO or other additives in the buffer?
Small amounts of DMSO (typically ≤2%) are tolerated, but higher concentrations may interfere with heat signals. Please inform us of all additives in advance so we can assess compatibility.
