- Protein quantification by UV absorbance at 280 nm is a direct, non-destructive method based on the intrinsic absorption properties of aromatic amino acids. This technique relies primarily on tryptophan and tyrosine residues, with minor contributions from phenylalanine and disulfide bonds between cysteine residues.
- The method follows the Beer-Lambert law, which states that absorbance is directly proportional to protein concentration and path length. The extinction coefficient of the protein, which depends on its amino acid composition, is crucial for accurate quantification. Standard extinction coefficients are available for many proteins, or they can be calculated based on amino acid sequence.
- A significant advantage of UV absorption at 280 nm is its non-destructive nature, allowing sample recovery after measurement. This feature makes it particularly valuable when working with precious samples or when the protein solution needs to be used for subsequent experiments. The method requires minimal sample volume, especially with modern micro-volume spectrophotometers.
- The technique’s main limitation is its dependence on aromatic amino acid content, particularly tryptophan and tyrosine. Proteins lacking these residues will show weak absorption, leading to unreliable measurements. Additionally, the presence of nucleic acids, which absorb strongly at 260 nm with overlap at 280 nm, can interfere with measurements.
- To account for nucleic acid interference, the Warburg-Christian method uses absorbance measurements at both 260 nm and 280 nm. This correction helps estimate protein concentration more accurately in samples containing nucleic acids. The ratio of A260/A280 also serves as a useful indicator of sample purity.
- Sample preparation is minimal, requiring only appropriate dilution to stay within the linear range of measurement (typically 0.1-1.0 absorbance units). The buffer used should have low absorption at 280 nm, and proper blanking with the same buffer is essential for accurate measurements.
- Modern instrumentation has greatly improved the convenience and reliability of UV absorption measurements. Micro-volume spectrophotometers can measure samples as small as 0.5-2 μL, making the technique particularly valuable when sample volume is limited. These instruments often include built-in path length correction and software for various calculations.
- The method’s accuracy depends on several factors, including proper instrument calibration, sample clarity, and accurate knowledge of the protein’s extinction coefficient. Turbidity or the presence of light-scattering particles can lead to overestimation of protein concentration. Centrifugation or filtration may be necessary to remove particulates.
- Temperature and pH can affect UV absorption measurements, although these effects are generally minor. Maintaining consistent measurement conditions and using appropriate buffer systems help ensure reproducible results. Regular instrument calibration and validation using standard proteins are recommended.
- For unknown proteins or complex mixtures, an average extinction coefficient can be used, though this introduces some uncertainty. A commonly used approximation is that a 1 mg/mL protein solution has an A280 of approximately 1.0, but this can vary significantly depending on the protein’s composition.
- Documentation of measurement conditions, including buffer composition, dilution factors, and any correction factors applied, is important for reproducibility. Regular verification of instrument performance using standard solutions helps maintain measurement accuracy over time.
- The technique’s simplicity, speed, and non-destructive nature make it a valuable tool in protein research, particularly when combined with other quantification methods for validation. Its limitations can often be overcome by using complementary techniques or appropriate correction factors.
- Quality control measures should include regular blank measurements, multiple readings of each sample, and verification of measurements using different dilutions to ensure linearity. Proper sample handling and storage conditions help maintain protein stability and measurement accuracy.
