Protein Concentration Calculator

Measuring protein concentration at 280 nm exploits the fact that only three residues absorb appreciably in the near ultraviolet: tryptophan, tyrosine, and cystine formed from disulfide-bonded cysteines. Their combined absorbance obeys the Beer-Lambert relation A equals epsilon times path length times molar concentration, so concentration is A divided by the product of epsilon and l. Because epsilon depends entirely on how many of those residues a given protein carries, it is not a universal constant: bovine serum albumin has an epsilon near 43,800 per M per cm and an average of 66,500 daltons, while an immunoglobulin G near 150,000 daltons runs above 200,000 per M per cm. Multiplying molar concentration by molecular weight and dividing by 1,000 converts to mg/mL, and the dilution factor restores the stock value. The practical window is narrow. Readings should fall between roughly 0.1 and 1.0 absorbance units in a 1 cm cuvette, since below that the difference between incident and transmitted light is swamped by noise and above it stray light and detector nonlinearity distort the result. Several artifacts inflate A280 specifically. Nucleic acid contamination absorbs strongly at 260 nm with a shoulder at 280, and an A260 over A280 ratio above about 0.6 signals it. Imidazole carried over from nickel affinity purification, DTT, and some detergents absorb in the same region. Light scattering from aggregates or particulates raises apparent absorbance across the spectrum and can be partly corrected by subtracting a 320 or 340 nm reading. A protein with no tryptophan and few tyrosines has a very small epsilon and is effectively invisible to this method, which is when Bradford, BCA, or a fluorometric assay is used instead.

Ultraviolet absorbance became a protein assay after the aromatic amino acids were shown to account for the 280 nm band. In 1942 Otto Warburg and Walter Christian published the nucleic acid correction that bears their name, using the ratio of absorbance at 260 and 280 nm to estimate protein in the presence of nucleotide contamination; their equations remained standard in enzymology for decades. The method spread rapidly once commercial instruments made the measurement routine, above all the Beckman DU spectrophotometer introduced by Arnold Beckman in 1941, which brought reproducible quantitative UV readings within reach of ordinary biochemistry laboratories. The weak point was the extinction coefficient, which had to be determined experimentally for each purified protein, typically by dry weight or nitrogen analysis. That changed once sequences became available. Building on earlier work by Herbert Edelhoch in 1967, C. Nick Pace and colleagues published a systematic analysis in Protein Science in 1995 showing that epsilon at 280 nm can be predicted from sequence with a few percent accuracy by summing contributions of about 5,500 per M per cm per tryptophan, 1,490 per tyrosine, and 125 per cystine. The Swiss Institute of Bioinformatics implemented this in the ProtParam tool on the ExPASy server, making calculated extinction coefficients freely available, and microvolume instruments introduced around 2000 reduced the sample requirement from a full cuvette to about one microliter, which is why A280 remains the fastest and most nondestructive protein quantification in routine use.