Is your sample a purified protein? Purified protein samples can be accurately measured using direct absorbance at 280 nm. Absorbance at 280 nm is mostly due to the aromatic chains on the amino acids Tryptophan (Trp) and Tyrosine (Tyr). Protein A280 is the most popular quantification method because it is fast and simple, requires no reagents or standard curves, and consumes very little sample. Using the absorbance at 280nm (A280), protein concentration (c) is calculated using the Beer-Lambert equation A280 = c * ε * b (ε is the wavelength-dependent protein extinction coefficient, b is the pathlength). Each pure protein has a unique extinction coefficient. For accurate results, the correct protein extinction coefficient ε must be entered or the closest Sample Type must be selected. The NanoDrop One Protein Editor feature allows you to save the extinction coefficients of specific proteins so that you can customize your Sample Type options.
In addition to concentration, protein measurements on the NanoDrop One spectrophotometer deliver information about contaminants in the sample. The NanoDrop One Thermo Scientific™ Acclaro™ Sample Intelligence Technology uses mathematical algorithms to detect nucleic acids in protein samples and correct the concentration result as needed. Sample purity can also be assessed by looking at the A260/A280 value. An A260/A280 value >1 may indicate nucleic acid contamination in the protein sample.
Proteins in complex mixtures such as cell extracts or lysates are best measured using a protein colorimetric assay such as Bradford, BCA, Lowry or Thermo Scientific™ Pierce™ 660nm Assay. These assays provide protein-specific concentrations, avoiding absorbance from cell components that absorb in the UV range and would inflate A280. For buffer compatibility with each assay, check the manufacturer’s product literature. The above colorimetric assays are preconfigured applications on the NanoDrop One/OneC, NanoDrop 2000/2000c and NanoDrop 8000 instruments. (Table 2)