An Overview of Protein Assay Methods

Proteins are one of four macromolecule building blocks that are necessary for any form of life. That’s why being able to analyze proteins with a protein assay is crucial in both research and diagnosis.

Total protein assay kits can be used to determine the total protein concentration of a sample.

To help you choose the right method to measure your protein, read this review of the main protein quantification methods.

Bicinchoninic Acid (BCA) Assay

This colorimetric, two-step protein concentration assay was first developed in 1985. It is relatively young compared to others, such as the Lowry assay.

First, complex the protein by using copper ions. Next, the copper that is now bound with the protein will give off a vibrant purple hue. The shade of purple corresponds to the amount of protein in the sample.

Then, compare the sample to the standard curve to determine the protein.

The BCA assay is a good choice if your protein sample has less than 5% detergents. As this method relies on copper, certain chemicals can interfere with the BCA assay, such as ammonia.

Bradford (Coomassie) Assay

Bradford Assays bind the protein to Coomassie dye in acidic conditions. As a result, the color changes from brown to blue.

When the Coomassie dye binds to the amino acids in the protein, it becomes blue and absorbs at 595 nm. The absorption in your sample can then be compared to a standard curve to determine the protein concentration.

This method is simple, fast, and stable for around 60 minutes. Keep in mind that it only works on proteins larger than 3 kDa. Unlike the BCA, the Bradford is sensitive to certain detergents, such as SDS and Triton X-100.

Folin-Lowry

The Folin-Lowry is a reliable and time-tested protein quantification that dates back to 1951. This has two steps.

Step one: complex copper with nitrogen found in your protein. Step two: the now complex tyrosine and tryptophan will react with the Folin-Ciocalteu phenol reagent to give off a greenish blue shade that absorbs at 650-750 nm.

The great thing about this assay is that the results are stable. So you can estimate how much protein is in your sample by comparing it with a previous standard curve.

The downside of this assay is that it is incompatible with many chemicals and reducing agents.

HPLC (High-Performance Liquid Chromatography)

In a liquid chromatography protein concentration assay, the sample is transported through an absorbent column by a solvent at high pressure.

Each amino acid side chains will react differently. The ones that react least will move down the column more quickly than the side chains that react the most.

As fractionated proteins flow out of the column, each fraction is identified either by UV spectrometry or by mass spectrometry. A dye can be used to make detection easier.

Final Thoughts on Protein Assay

Thanks for reading. We hope you found this list of the various commonly used protein assay quantification methods useful.

Next, check out ProDM, a kit for tryptic digestion monitoring for a successful shotgun proteomics.