Question

Why is the Bradford Assay a superior method for determining protein concentration compared to other protein concentration methods such as taking an absorbance at 280 nm?

Why might your values be different from those of the manufacturer?

Answer 1

Absorbance at 280 nm :-

How it works: Aromatic residues, like tyrosine and tryptophan, absorb UV light at 280 nm. So, if you have an extinction coefficent for your protein (e), you can measure the absorbance in a UV/Vis spectrometer and calculate the concentration of your protein using Beer’s law (A = elc, where l is the path length of the spectrometer).

Because ProtParam only considers the linear sequence of your protein and doesn’t take into account the structure, which can affect the extinction coefficient, you’ll want to denature your protein before you measure the absorbance. (I like to denature proteins in 6 M guanidinium.)

Advantages: This technique is quick and doesn’t require any special reagents, except for the guanidinium, which you may have on hand anyway.

Disadvantages: This method relies on having an accurate extinction coefficient for your protein, which depends on the number of aromatic residues. If there aren’t a decent number of aromatic residues, your extinction coefficient will be quite low, and you will need a fairly concentrated sample to get a reasonable absorbance (generally an absorbance between 0.1 and 1.0 is considered within the “linear range”). Also, ProtParam warns that there may be at least a 10% error in the extinction coefficient if there are no tryptophans in your protein. Therefore, if your extinction coefficient is low, which is likely the case if there are no tryptophans in the sequence, a 10% error could significantly throw off your assessment of the final protein concentration.