In my various laboratory experiences (N=5; probably not enough to achieve any kind of statistical significance) it seems that labs either use a Bradford or a BCA protein assay to quantify protein concentrations.
Both assays come in convenient kits that can be performed in micro, multiwell plates or in a standard, larger volume format.
So how does a lab choose?
Bradford protein assay
The Bradford assay is based on the ability of Coomassie blue (you know, they dye you use to stain your protein gels) to bind protein causing the dye to shift from a red color to a blue color. This shift can be quantified by measuring the absorbance of your samples at 595 nm.
The Bradford assay is quick – samples can be read 5 minutes after the addition of the dye to your sample.
A lot of people like the Bradford assay because fewer substances interfere with the assay (including reducing agents) – with the notable exception of high concentrations of some detergents. Which can be a problem if you require a lot of detergent to lyse your cells.
In addition, sometimes your samples need to be diluted before measuring them with Bradford leading to final calculation confusion.
Warning – don’t plan on washing and reusing your cuvettes. The Bradford reagent stains the plastic.
BCA (bicinchoninic acid) protein assay
The BCA assay is based on the traditional Lowry assay except bicinchoninic acid is used resulting in a very pretty purple color that absorbs at 560 nm.
BCA is sensitive and has a broad dynamic range – capable of measuring protein concentrations of 0.5 μg/mL to 1.5 mg/mL
A depressing list of things can interfere with BCA, including, carbohydrates, catecholamines, tryptophan, lipids, phenol red, cysteine, tyrosine, impure sucrose or glycerol, uric acid, iron and hydrogen peroxide – but truthfully I have never found this to be a problem with general samples in the lab.
BCA is not compatible with reducing agents and you also have to invest some time in the assay as samples are read after 30-60 minutes of incubation.
Based on this information though, I wonder – why should a lab choose?
Why not use the Bradford for a quick assay to measure less concentrated proteins, or those in buffers containing reducing agents and turn to the BCA for highly concentrated samples lysed in detergent?
We are curious – do you prefer one assay over the other?
Take the poll to tell us what you use and leave us a comment to tell us why.
Photo courtesy of West Point – The U.S. Military Academy