Protein quantification
Coomassie G-250 binds arginine and aromatic residues and shifts from red-brown to blue, read at 595 nm within minutes. It is fast and sensitive and tolerates reducing agents, but the response varies about twofold between proteins and is distinctly non-linear, and detergents interfere.
Our ruleSimulations prepare you for the bench but don't replace hands-on practice.
Bradford
Coomassie G-250 binds arginine and aromatic residues and shifts from red-brown to blue, read at 595 nm within minutes. It is fast and sensitive and tolerates reducing agents, but the response varies about twofold between proteins and is distinctly non-linear, and detergents interfere.
How it differs from the standard method
- IgG gives less than half the colour of the same mass of BSA, so an antibody read against a BSA curve comes out at roughly half its real concentration.
- Read 5–10 min after adding the reagent; the complex needs time to form, and the dye then slowly precipitates on the plastic.
- The curve flattens towards the top of the range: interpolate or fit a curve rather than drawing a straight line.
- SDS and Triton X-100 bind the dye themselves, raising the blank and distorting the curve; detergent-compatible formulations exist but should be validated.
- Coomassie stains cuvettes and plates; use disposable ones.
1Every protein assay counts something other than protein
There is no reagent that responds to “protein”. Each assay responds to some feature that proteins have in varying amounts, and converts it into a mass by comparison with a protein whose mass is known.
2Bradford: fast, sensitive, and protein-dependent
Coomassie Brilliant Blue G-250 in acid is a brownish red. When it binds protein it shifts to its blue anionic form, absorbing at 595 nm, within a few minutes.
3BCA and Lowry: copper chemistry that counts peptide bonds
In alkaline solution, peptide bonds reduce Cu²⁺ to Cu⁺ (the biuret reaction), and cysteine, tyrosine and tryptophan add to it. BCA then chelates the Cu⁺ into an intense purple complex read at 562 nm; Lowry instead uses the Cu⁺ to reduce the Folin–Ciocalteu reagent to a blue product.
Arginine and aromatics
Five minutes, very sensitive, reducing agents fine. Detergents interfere and the answer depends on the protein, sometimes by twofold.
Peptide bonds
The default for lysates: detergents tolerated, response more uniform across proteins. Thiols and chelators interfere; 30 min at 37 °C.
Peptide bonds, then Folin
The older copper assay with more interferences and more timed steps. Comparable to BCA in what it measures.
Tryptophan and tyrosine
No reagent, no standard, nothing consumed. Needs the protein's extinction coefficient and useless for a protein with no aromatic residues.
4A280: the method that gives the sample back
Tryptophan and tyrosine absorb ultraviolet light near 280 nm, and cystine weakly, so the absorbance of a pure protein follows Beer–Lambert: A = ε × c × l. The extinction coefficient can be calculated from the sequence, and it varies widely, a 1 mg/mL solution gives about 0.67 for albumin and 1.4 for IgG, so the convenient assumption that an absorbance of 1.0 means 1 mg/mL is wrong by tens of percent in both directions.
5Running any of them well
- Make the standards in the same buffer as the samples, and blank against that buffer. Matrix effects shift the whole curve and cannot be corrected afterwards.
- Use a standard that resembles the sample: BSA for lysates by convention, IgG for antibodies, ideally the purified protein itself quantified independently.
- Read samples inside the range of the standards. When samples span more than the assay's range, assay each at two dilutions rather than extrapolating.
- Interpolate between standards, or fit a curve; do not force a straight line through a curved response.
- Run replicates. A single well cannot tell a pipetting error from a real difference.
- Report which assay and which standard you used. A protein concentration without them cannot be compared with anyone else's.
Key terms
Practise these as flashcards →Absorbance measured for a dilution series of a protein of known concentration, used to convert a sample's absorbance into an equivalent mass of that standard protein.
How much signal a given protein produces per microgram relative to the standard. Bradford response factors vary about twofold between common proteins; copper-assay ones much less.
The absorbance of a protein at a defined concentration and path length, calculable from its tryptophan, tyrosine and cystine content. Often quoted as the A280 of a 1 mg/mL solution in a 1 cm path.
A change in the assay's response caused by the sample's buffer rather than its protein. The reason standards and blanks are made in the sample buffer.
A buffer component the assay responds to as if it were protein, or that suppresses the response: detergents in Bradford, reducing agents and chelators in BCA, aromatic detergents at 280 nm.
The span of concentrations over which an assay's standard curve is usable. Samples outside it are diluted or concentrated, not extrapolated.
The distance light travels through the sample. 1 cm in a standard cuvette; in a plate it is the depth of liquid, about 0.6 cm for 200 µL in a 96-well plate.
Light deflected by aggregates or particles that never reaches the detector, which an absorbance reader cannot distinguish from absorbance.
When to use it
- Normalising lysates before SDS-PAGE and a western blot, so that a difference in band intensity is not just a difference in how much was loaded.
- Checking yield and concentration between steps of a purification, where A280 keeps the sample and a colorimetric assay would consume it.
- Setting up an enzyme or binding assay that needs a known molar concentration of a purified protein, where the choice of method and standard decides the accuracy of every constant you report.
- Measuring protein in a buffer that rules out one method: BCA for detergent lysates, Bradford for samples containing reducing agent, A280 only for pure protein.
Compared with alternatives
The underlying measurement; go there for Beer–Lambert, stray light and the limits of an absorbance reading.
The instrument most protein assays are read on, and the source of the path-length and linearity issues they share.
The step before, and the reason the buffer decides the assay: a RIPA lysate rules out Bradford, a reducing lysis buffer complicates BCA.
A Coomassie-stained gel against a known standard is the independent check when two assays disagree about a purified protein.
The nucleic acid equivalent, with the same lesson: absorbance is fast and non-destructive, and a dye assay is more specific.
Sub-techniques
Choosing one here switches the whole page, including the protocol and the lab, to it.
Coomassie G-250 binds arginine and aromatic residues and shifts from red-brown to blue, read at 595 nm within minutes. It is fast and sensitive and tolerates reducing agents, but the response varies about twofold between proteins and is distinctly non-linear, and detergents interfere.
- IgG gives less than half the colour of the same mass of BSA, so an antibody read against a BSA curve comes out at roughly half its real concentration.
- Read 5–10 min after adding the reagent; the complex needs time to form, and the dye then slowly precipitates on the plastic.
- The curve flattens towards the top of the range: interpolate or fit a curve rather than drawing a straight line.
- SDS and Triton X-100 bind the dye themselves, raising the blank and distorting the curve; detergent-compatible formulations exist but should be validated.
- Coomassie stains cuvettes and plates; use disposable ones.