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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|
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
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.
The Bradford assay is a colorimetric assay that relies on the binding of protein to Coomassie Brilliant Blue.