Bradford BCA and absorbance at 280 nm

A number written on a tube of protein is only as good as the chemistry that produced it. Bradford dye binding, a bicinchoninic acid copper assay, and absorbance at 280 nm can all be called protein quantification, and they do not answer the same question. One is a colour from a dye, one is a colour from reduced copper, and one is ultraviolet light absorbed by a few side chains. The decision this page supports is which of those readings you are allowed to use for the buffer in front of you, and what you must write beside the number so the next person can repeat it. The companion on how laboratories estimate protein concentration sets the wider frame. A gel, covered in reading a protein gel, still has to show whether that mass is one polypeptide.

What each reading is actually made of

The Bradford assay, introduced as a protein-dye method by Bradford in 1976, uses Coomassie dye. Free dye and protein-bound dye absorb light differently, so the well changes colour as protein binds. Binding is not a fair census of peptide bonds. Arginine, and to a lesser extent lysine, histidine and some aromatic residues, contribute strongly. A basic protein and a small acidic protein of equal mass can give unequal colour. Immunoglobulin and albumin are a practical pair that often disagree. That protein-to-protein variation is a property of the chemistry, not a pipetting error.

Detergent is the other Bradford problem. Sodium dodecyl sulfate, Triton-type detergents and some other surfactants disturb the dye equilibrium. The direction and the size of the error depend on the formulation and the concentration. A lysate prepared for electrophoresis, with SDS already in it, is a poor candidate for a classical Bradford reading. Some commercial versions raise the detergent tolerance. That claim belongs to the bottle you bought. It does not transfer to a different Coomassie recipe.

The BCA assay, described by Smith and colleagues in 1985, is a copper method. In alkaline solution, peptide bonds and some side chains reduce Cu2+ to Cu1+. Bicinchoninic acid then forms a coloured complex with the reduced copper. Because the peptide backbone contributes, the protein-to-protein spread is often narrower than with Coomassie, though it does not disappear. BCA formulations are widely chosen when the sample must contain detergent, because many detergents that ruin a Bradford plate are tolerated here. The trade is reductant. Dithiothreitol, beta-mercaptoethanol and related reducing agents reduce copper themselves, so they invent colour. Chelators such as EDTA can pull copper out of the reaction. A high concentration of a reducing sample buffer is a reason to change the assay or to remove the reductant first, not a reason to subtract a guessed blank.

Absorbance at 280 nm does not add a dye or a metal. Tryptophan, tyrosine and cystine account for almost all of the near-ultraviolet absorption of a typical polypeptide. A pure protein, a 1 cm path, and an extinction coefficient that matches the construct convert absorbance into concentration. Nucleic acids absorb strongly in the same region and inflate the number. The ratio of absorbance at 260 nm to absorbance at 280 nm is the usual warning: protein-rich samples sit lower than nucleic-acid-rich samples. Imidazole and some buffers also absorb. A water blank under a sample dissolved in urea or a coloured component is not a blank. The assumptions packed into a calculated coefficient are set out in what an extinction coefficient assumes.

The standard is part of the result

Bovine serum albumin is common because it is available and usually well behaved in these assays. It is not your protein unless you are measuring albumin. A Bradford number read against albumin and reported as if it were milligrams of your enzyme hides the dye-binding difference. Say albumin equivalents when that is the scale you used. If you later switch to a standard made from the purified protein itself, expect the number to move. Both readings can be careful. They are not interchangeable in a table.

For a pure protein with a known coefficient, A280 avoids that translation step. For a crude lysate you will load on a gel, an albumin-equivalent dye or copper assay is an operational load, not a molarity of the enzyme you care about. Inclusion-body material dissolved in urea or guanidine needs an assay the denaturant does not break. High denaturant often pushes people toward A280 with a matched blank, once a coefficient exists. Check the interference notes for the reagent generation you have. Public method notes on protocols.io are useful for seeing where laboratories place these assays. They are not a substitute for the insert in the box.

A path with branch points

Start from the additives, not from the assay the bench next door prefers. If the sample contains SDS or another strong detergent and little reductant, a BCA-type copper assay is usually the candidate to test. If the sample is a simple salt buffer and you can accept protein-to-protein variation, Bradford is often the faster colour. If the sample is a single known polypeptide and nucleic acid is low, measure A280 and record the coefficient and the path length.

Then look at the blank. A blank that already looks as blue or as purple as the standards means the buffer is creating signal. Stop and identify the additive. Do not subtract an overflowing blank and call the remainder a concentration. If standards are flat, the reagent may be old, the incubation may be wrong, or a chelator may have removed the copper. Follow the manufacturer protocol for time and temperature. Do not invent a microlitre table from memory or from a different kit.

If the unknown is darker than the top standard, dilute it in the same buffer and read again. A value past the curve is a prompt to dilute. If Bradford and A280 disagree on a pure protein, believe the disagreement. It is information about composition, dye response, or a contaminant. Run the sample on a gel before you average the two numbers into a false compromise.

Readings that look precise and are not

A spectrophotometer can print three decimal places for a cloudy sample. Scatter is not absorption by tryptophan. Spin or filter first, and read the cleared liquid, as the aggregation note in this cluster describes. A plate reader with a short path does not automatically match a 1 cm coefficient. Either use the instrument's path-length correction as the manufacturer defines it, or stay with a relative standard curve and say so.

Old Coomassie reagent and a copper reagent that has sat warm can both drift. A standard curve that no longer resembles last month's curve is a failed control. Make a fresh curve. Do not apply last month's slope to today's wells. If duplicates scatter, suspect mixing, bubbles, or a sample that is still settling, before you suspect the biology.

Handling, research use and the room

Coomassie reagent, alkaline copper reagent and the acids used around them are chemical hazards. Follow the safety note for the formulation you have. This page is a research explainer. It is not a clinical protein method and not biosafety approval. Your institution decides containment for the lysate.

In a hot, humid laboratory, "room temperature incubation" is not a temperature from a temperate kit insert. Keep the incubation in the range the protocol states, and do not leave a dye reagent in direct sun on a bench. A spectrophotometer that rebooted after a power cut needs a lamp warm-up and a standard check before a precious dilution series is trusted. Weigh hygroscopic standards with the same care you would give any other salt that takes up water. Record the assay, the standard, the dilution and the buffer. Those four words stop a later argument about whose number was right.

What to put in a reagent enquiry

Name the sample matrix first: lysate, elution, inclusion-body solubilisation, or a polished pool. Name the detergents and reductants at the concentrations you actually use. Say whether you need albumin equivalents for gel loading or a molarity tied to a coefficient. Reagents and chemicals is the catalogue family for assay components and buffer salts. A quote can carry that matrix description. The custom protein expression and purification page is an enquiry reference if the quantification sits inside a larger expression question. It is a place to discuss a method. It is not a statement that a purification is already being run.