Colorimetric or Copper Based Protein Assays - How to Decide Which is Best

While protein assays have a variety of uses in life science research, there is no single assay method that is suitable for all applications.  Despite all the advancements in modern science, a protein assay method that is not affected by any non-protein components or by the differences in protein composition does not exist. For this reason, protein laboratories find it necessary to have more than one type of protein assay for research applications.

Each assay has its own advantages and limitations. Thus, if you want to get accurate results from your experiment, you should be able to select the most suitable assay for your application.

Types of Colorimetric Protein Assays

Based on the chemistry involved, the two most common methods for the colorimetric detection and quantitation of total protein are the protein-dye binding assay and/or copper ion-based chelation assay.

Dye Binding Assays

Dye-binding assays, such as the Bradford (Coomassie) and 660nm protein assays, are based on the binding of protein molecules to Coomassie dye under acidic conditions. Once the protein molecules bind to the dye, the color shifts from brown (Amax= 465nm) to blue (Amax= 610nm). The change in color density (which is read at 595nm) is proportional to protein concentration.

The basic amino acids (arginine, lysine and histidine) play a vital role in the formation of dye-protein complexes and the resulting spectral shift while smaller proteins (less than 3kDa) and amino acids do not produce color changes.

Copper Ion Based Assays

In copper ion based protein assays, the protein solution is mixed with an alkaline solution of copper salt to facilitate the chelation of cupric ions (Cu²⁺) with the peptide bonds, and the subsequent reduction of the cupric ions (Cu²⁺) to cuprous ions (Cu⁺). Any excess copper ions will remain unbound to the peptide bonds and will be available for detection.

Protein assays based on copper ions can be divided into two groups - (1) assays that detect reduced cuprous ions (Cu⁺) and (2) assays that detect unbound cupric (Cu²⁺) ions. Bicinchoninic acid (BCA) or Folin Reagent (phosphomolybdic/ phosphotungstic acid) may be used to detect cuprous ions. Upon the reduction of the reagent used, a blue color is produced. The amount of color produced can be read at 650nm to 750nm and is proportional to the amount of peptide bonds.

Note: The presence of tyrosine, tryptophan, cysteine, histidine and asparginine in protein enhances the resulting color density.

In assays based on the detection of unbound cupric ions, alkaline copper is mixed with the protein solution and the unchelated cupric ions are then detected with a color-producing reagent that reacts with cupric ions. The amount of color produced is inversely proportional to the amount of peptide bond in the sample. 

Selecting Protein Assays: Some Factors to Consider

There are a number of factors that you should consider when choosing an assay.  Here are some of them.

• Compatibility with the sample type and components. The nature of the protein sample and the presence of non-protein agents in protein solutions can significantly affect the results of your experiment. Keep in mind that the presence of reducing agents, metal chelating agents, dyes, amines, and sugars interfere with copper based protein assays while protein solutions containing detergents interfere with the dye based protein assays.
• Assay range and required sample volume. Most colorimetric assays require at least 0.5µg proteins for a reliable estimation. Thus, for hard to obtain samples, methods that require the least amount of sample for a reliable estimation should be considered.
• Protein-to-protein uniformity. Dye based protein assays and those involving the reduction of cuprous ions to cupric ions have significant protein-to-protein variation.
• Time considerations.  The complexity of the sample and the chosen assay method dictate the amount of time you need to perform your protein assay. Protein samples containing interfering agents and assays that use standard plots or curves will consume more time.
• Instrumentation requirements. The availability of spectrophotometer or plate reader necessary to measure the color produced (absorbance) by the assay may also affect your choice.