Protein quantitation and protein assays are critical for accurately determining the protein concentration in a sample. There are a variety of protein quantitation methods including UV absorbance assays, reagent-based assays, and immunoassay technologies. Each of these protein quantitation technologies has unique benefits and the suitability of the assay depends on the sample type and/or volume that is available for analysis. For example, some of the dye-based assays may interfere with chemicals found in buffer preparations and an alternative assay may be more appropriate. Scientists need to consider the limitations of each assay to determine which is the best option for their sample.
Using ultraviolet (UV) absorbance to measure protein concentration is a relatively simple protein quantitation assay. Amino acids with aromatic side chains (tryptophan, tyrosine, etc.) provide proteins with their distinctive UV absorbance at 280 nm. Because these amino acids absorb UV light at 280 nm, the absorbance at this particular wavelength can be obtained through a spectrophotometer and used to estimate protein concentrations in samples. This relatively quick assay is frequently used in laboratories and the Warburg-Christian method is typically performed for the protein concentration estimation. However, using UV absorbance for protein concentration may have high variability because non-protein components in a sample may interfere with absorbance measurements. Additionally, mixtures with different proteins in a sample could cause varying absorbance readings due to the difference in amino acid compositions.
Reagent-based assays overcome the compatibility issues that are observed with UV absorbance methods. Examples of reagent-based assays for protein quantitation include those that utilize colorimetric methods, such as bicinchoninic acid (BCA), Lowry, and Bradford assays. The BCA method and the Lowry method both involve the formation of a copper-protein complex. These are sensitive assays and are less variable than the Bradford assay. However, the Bradford assay is rapid, easy to perform, and is compatible with certain reducing agents, unlike the BCA and Lowry assays.
Some assays may not be able to support accurate protein quantitation if there are multiple proteins in a sample or quantities are below the detection threshold. Powerful immunoassay technologies, using various detection methods, provide an alternate method to precisely quantitate proteins from a variety of sample types. For example, multiplex assays allow researchers to quantitate multiple proteins in a sample simultaneously. In addition, single molecule counting technology can measure femtogram levels of proteins to help identify and quantify low-level proteins in small samples. Research applications using these technologies include measuring biomarkers in healthy tissues or those associated with disease progression to better understand certain disease states.