How buffer conditions and sample composition can influence your results.
Protein purification is rarely a one-buffer affair. As your sample moves through lysis, capture, and elution steps, it encounters a series of changing conditions — each of which can impact the accuracy of your quantification method.
Getting a reliable readout during purification isn’t just about the concentration — it’s about the context.
Common interferences to look out for
Some purification buffers and additives can interfere with detection, especially in surface- or chemistry-based assays. These include:
- Imidazole – Often used in His-tag elution buffers; can interfere with metal-binding or redox surfaces
- High-salt buffers – May affect protein binding, surface interaction, or signal generation
- Detergents and surfactants – Can denature proteins, block binding sites, or alter enzyme kinetics
- Reducing agents (e.g. DTT, TCEP) – May disrupt redox reactions depending on the assay chemistry
These components don’t always cause failure — but they can reduce sensitivity, introduce variability, or lead to signal suppression.
Best practices for better results
To ensure consistent and interpretable readings during purification:
- Dilute elution fractions when buffer components are known to be incompatible
- Use matched standards prepared in similar buffer conditions
- Pre-clear samples to remove particulates or aggregates
- Track buffer composition at each step — don’t rely on assumed compatibility
- Run blanks and controls alongside test samples when working with unknown matrices