Protein Quantitation
Protein quantitation and protein assays are essential for accurately determining the concentration of proteins in a sample. There are a variety of protein quantitation methods available, including UV absorbance assays, reagent-based assays, and immunoassay technologies. Each of these techniques offers unique benefits, and the choice of the most suitable assay depends on factors such as the sample type and the available sample volume. For instance, some dye-based assays may be interfered with by chemicals present in buffer preparations, making an alternative assay more appropriate for certain applications. It is crucial for scientists to carefully consider the limitations of each assay in order to determine the best option for their specific sample and analysis needs,
Overview
UV Absorbance Assays
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
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.
Immunoassays
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.