How to Measure Protein Concentration Using Absorbance at 280 nm

Measuring protein concentration is a fundamental task in biochemistry, essential for various applications ranging from enzyme assays to structural biology. One common method to estimate protein concentration is by measuring absorbance at 280 nm using a spectrophotometer. This technique is favored for its simplicity and speed, and it relies on the intrinsic absorbance properties of aromatic amino acids, particularly tryptophan and tyrosine.

The principle behind using 280 nm absorbance involves the presence of aromatic rings in the tryptophan and tyrosine residues, which absorb ultraviolet light at this wavelength. The absorbance is directly proportional to the concentration of these amino acids, and by extension, the protein concentration. Here's a detailed guide on how to measure protein concentration using this method.

**Sample Preparation**

Begin by preparing a protein solution in an appropriate buffer. It is important to ensure that the buffer itself does not absorb significantly at 280 nm, as this could interfere with the measurement. Typical buffers like phosphate-buffered saline (PBS) or Tris buffer are suitable choices. The concentration of the protein should ideally fall within the linear range of the spectrophotometer used, usually between 0.1 to 1.0 mg/mL. If the concentration is too high, consider diluting the sample to bring it within this range.

**Calibrating the Spectrophotometer**

Before measuring your protein sample, calibrate the spectrophotometer using a blank solution, which contains the buffer without any protein. This step is crucial as it sets a baseline absorbance, accounting for any absorbance contributions from the buffer itself. After calibration, the spectrophotometer should read zero absorbance for the blank solution.

**Measuring Absorbance**

Pour the protein solution into a clean cuvette, ensuring that the cuvette is free from scratches or residues that could affect the absorbance reading. Insert the cuvette into the spectrophotometer and record the absorbance at 280 nm. It is advisable to take multiple readings and use the average value to enhance the accuracy of your results.

**Calculating Protein Concentration**

The absorbance at 280 nm can be used to estimate protein concentration through the Beer-Lambert Law, which states that absorbance (A) is equal to the product of the molar absorptivity (ε), the path length of the cuvette (l), usually in centimeters, and the concentration of the solution (c):

A = εlc

For proteins, the molar absorptivity at 280 nm is typically determined experimentally and can vary depending on the specific protein composition. As a general guideline, an average value for ε can be used if the exact composition is unknown, although this will reduce the accuracy of the concentration estimate. Alternatively, if the amino acid composition of the protein is known, ε can be calculated more precisely.

**Considerations and Limitations**

While absorbance at 280 nm is a convenient method for estimating protein concentration, it has limitations. Proteins lacking tryptophan and tyrosine residues will not absorb strongly at this wavelength, leading to inaccurate concentration estimates. Furthermore, the presence of nucleic acids, detergents, or other absorbing substances in the solution can interfere with the measurement. In such cases, alternative methods such as the Bradford assay, BCA assay, or Lowry assay may be more appropriate.

Additionally, care must be taken to ensure that the protein is completely dissolved and homogeneously distributed in the buffer, as undissolved particles can scatter light and artificially inflate absorbance readings.

In summary, measuring protein concentration using absorbance at 280 nm is a quick and efficient technique suitable for many applications. By carefully preparing samples, calibrating equipment, and considering the limitations of the method, reliable protein concentration estimates can be obtained, aiding in the successful execution of biochemical experiments.