Total protein quantification using A280
Applications | 2020 | Unchained LabsInstrumentation
Protein quantification at 280 nm is a fundamental analytical technique in biochemical research and industry. It provides a rapid and label-free method to assess protein concentration, critical for applications such as enzyme kinetics, bioprocess monitoring and quality control.
This application note presents the use of the classic Protein (Turbidity) application on the Lunatic and Little Lunatic systems. The aim is to quantify total protein concentration based on the A280 peak height and compare performance against conventional methods.
Instruments:
Samples are measured using the Protein (Turbidity) application. A buffer blank corrects baseline absorbance. The A280 absorbance is converted to concentration (mg/mL) using the E1% extinction coefficient (default 10 g-1 L cm-1). Background turbidity is subtracted to isolate the protein spectrum. Purity is assessed via the A260/A280 ratio (~0.6 for pure proteins). Saturation and linear range warnings are indicated by red bands on the spectra.
The Lunatic platforms display results in overview and detail views, including A280 concentration and A260/A280 ratio. Report generation supports HTML, XML, TXT, CSV, and on Lunatic XLSX and PDF. In a case study with a BSA/IgG dilution series, Lunatic results closely matched those from a NanoDrop 2000, showing high linearity (R² near 1) and accuracy. Detergent compatibility tests indicated maximum non-interfering concentrations for common surfactants such as Tween 80 (10%), Triton X-100 (0.01%), Tween 20 (0.1%), NP40 (0.01%), SDS (0.5%) and CHAPS (20%).
Advancements may include expanded wavelength scanning, enhanced chip materials for greater sensitivity, integration with automated liquid handling, and AI-driven data analysis for improved throughput and decision support in proteomics and biomanufacturing.
The Lunatic and Little Lunatic systems offer robust, user-friendly platforms for total protein quantification by A280 absorbance. They deliver accuracy comparable to established instruments, with added benefits in throughput, reporting and sample versatility.
No references were listed in the source document.
Particle characterization, UV–VIS spectrophotometry
IndustriesProteomics
ManufacturerUnchained Labs
Summary
Significance of the Topic
Protein quantification at 280 nm is a fundamental analytical technique in biochemical research and industry. It provides a rapid and label-free method to assess protein concentration, critical for applications such as enzyme kinetics, bioprocess monitoring and quality control.
Objectives and Overview
This application note presents the use of the classic Protein (Turbidity) application on the Lunatic and Little Lunatic systems. The aim is to quantify total protein concentration based on the A280 peak height and compare performance against conventional methods.
Used Instrumentation
Instruments:
- Lunatic system (Unchained Labs) with UV/Vis detection at 280 nm and specialized chip technology
- Little Lunatic platform with integrated application for small-volume measurements
Methodology
Samples are measured using the Protein (Turbidity) application. A buffer blank corrects baseline absorbance. The A280 absorbance is converted to concentration (mg/mL) using the E1% extinction coefficient (default 10 g-1 L cm-1). Background turbidity is subtracted to isolate the protein spectrum. Purity is assessed via the A260/A280 ratio (~0.6 for pure proteins). Saturation and linear range warnings are indicated by red bands on the spectra.
Main Results and Discussion
The Lunatic platforms display results in overview and detail views, including A280 concentration and A260/A280 ratio. Report generation supports HTML, XML, TXT, CSV, and on Lunatic XLSX and PDF. In a case study with a BSA/IgG dilution series, Lunatic results closely matched those from a NanoDrop 2000, showing high linearity (R² near 1) and accuracy. Detergent compatibility tests indicated maximum non-interfering concentrations for common surfactants such as Tween 80 (10%), Triton X-100 (0.01%), Tween 20 (0.1%), NP40 (0.01%), SDS (0.5%) and CHAPS (20%).
Benefits and Practical Applications
- Rapid and reliable total protein quantification with minimal sample volume
- Integrated data processing and versatile reporting formats
- Real time purity assessment via A260/A280 ratio
- Flexibility to accommodate a range of sample buffers and detergents
Future Trends and Opportunities
Advancements may include expanded wavelength scanning, enhanced chip materials for greater sensitivity, integration with automated liquid handling, and AI-driven data analysis for improved throughput and decision support in proteomics and biomanufacturing.
Conclusion
The Lunatic and Little Lunatic systems offer robust, user-friendly platforms for total protein quantification by A280 absorbance. They deliver accuracy comparable to established instruments, with added benefits in throughput, reporting and sample versatility.
References
No references were listed in the source document.
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