How do I determine protein concentration?
The quickest way to estimate the amount of protein in solution is to use UV-vis to measure absorbance directly, but this is generally not very accurate or sensitive. Highly accurate quantitation of most proteins can be achieved using either a Bradford or bicinchoninic acid (BCA) assay.
The table below summarizes the main differences between these different methods.
| UV-vis / A280 | Bradford assay | BCA assay | |
| Accuracy | Low; usually used as an approximation | High; gives accurate quantitation | High; gives accurate quantitation |
| Speed | Very fast; < 1 min | Fast; 20 mins | 1 hr, 30 mins |
| Chemistry | Tryptophan and Tyrosine residues absorb at 280 nm | Binding of Bradford reagent to protein; complex absorbs at 595 nm | Proteins reduce Cu2+ to Cu+; BCA chelates Cu+; complex absorbs at 562 nm |
| Considerations | DNA/RNA can interfere with results if present in high quantities; nucleic acids absorb at 260 nm Will not detect peptides without Tryptophan and Tyrosine amino acids | SDS (>0.1 %) and other detergents should be avoided | EDTA (>10 mM) and other strong chelating agents should be avoided Reducing agents should be avoided |
UV-vis / A280
Protein concentration can be estimated by measuring the UV absorbance at 280 nm; proteins show a strong peak here due to absorbance from Tryptophan and Tyrosine residues (commonly referred to as A280). This can readily be converted into the protein concentration using the Beer-Lambert law (see equation below). This method is used mostly for a very rough estimation of concentration, e.g. to check the success of purification.
A = ɛcl
- A is the absorbance (e.g. A280)
- ɛ is the molar extinction coefficient, M-1cm-1 – this can be found from the literature
- c is concentration in M
- l is the path length in cm – the length of the cuvette / microplate
Bradford Assay
The Bradford assay is a colorimetric assay that relies on the binding of protein to Coomassie Brilliant Blue.
- When the dye binds to protein in an acidic medium, a shift in absorption maximum occurs from 465 nm to 595 nm, with a change in color from brown to blue.
- Unknown protein concentrations are estimated by reference to a standard curve generated from proteins of known concentration, typically bovine serum albumin (BSA)
BCA
The BCA assay is a colorimetric assay that utilizes the reduction of Cu2+ ions by proteins, and subsequent binding of BCA.
- The generated Cu+ ions form an intensely colored complex with the BCA reagent, that has a very strong absorbance at 562 nm
- Unknown protein concentrations are estimated by reference to a standard curve generated from proteins of known concentration, typically bovine serum albumin (BSA)