Determine Protein Concentration Using Microvolumes with the Agilent Cary 60 UV-Vis Spectrophotometer

Importance of the Topic

Accurate protein quantification is essential in biochemistry, molecular biology and pharmaceutical research. Rapid, reliable and nondestructive methods that require minimal sample volume help preserve precious samples, increase throughput and reduce operational costs.

Objectives and Study Overview

This application note demonstrates how the Agilent Cary 60 UV-Vis spectrophotometer equipped with an ultra-microvolume cuvette can accurately determine protein concentration directly from microliter samples. The goal is to showcase method linearity, sensitivity and ease of use without sample dilution.

Methodology and Instrumentation

The workflow uses bovine serum albumin (BSA) as a model protein and compares standard 10 mm path length cuvettes with 1.0 mm and 0.2 mm mirrored-cap ultra-microvolume cuvettes. Measurements are performed on Agilent Cary 60 UV-Vis (or Cary 50) instruments. Key components:

• Agilent Cary 60 UV-Vis spectrophotometer with WinUV software
• Ultra-microvolume cuvettes: 1.0 mm and 0.2 mm path length mirrored caps
• BSA protein standard

Main Results and Discussion

Photometric linearity is maintained up to 2.3 Abs with the 1.0 mm cap and 23 Abs with the 0.2 mm cap, corresponding to equivalent 10 mm path-length absorbances of 23 Abs and 115 Abs respectively. This enables direct measurement of BSA from 0 to 190 mg/mL without dilution. The 10 mm standard cuvette extends detection down to 0.001 mg/mL. The ultra-microvolume format minimizes carryover and allows sample recovery. Simple wipe-cleaning between runs ensures rapid turnaround.

Benefits and Practical Applications

• Nondestructive analysis of precious samples
• Wide dynamic range (0.001–190 mg/mL) without dilutions
• High accuracy and reproducibility
• Reduced sample and reagent consumption
• Compatibility with automated cell changers and temperature-controlled holders for enzyme kinetics

Future Trends and Potential Uses

Integration of microvolume UV-Vis spectroscopy into high-throughput screening and online process monitoring will expand its use in proteomics and bioprocessing. Advances in microfluidics and automation could further reduce sample needs and increase analytical speed.

Conclusion

The Agilent Cary 60 UV-Vis spectrophotometer combined with ultra-microvolume cuvettes provides a versatile, accurate and efficient platform for protein concentration determination. It offers exceptional linearity and sensitivity while conserving valuable samples, making it an ideal choice for modern labs.