IR-Based Protein Quantitation Surpasses Colorimetric Assay Quantitation And Results Are Independent Of Detergents, Reducing Agents And Analysis Time

Accurate protein quantitation in complex mixtures is crucial for obtaining reproducible biological and biochemical data for both upstream and downstream applications. Colorimetric protein assays are frequently used to determine protein concentration; however, the calculated concentration can vary depending on the sample composition, sequence of the protein, and the time point within the chemical reaction at which the data are collected. Studies involving cell lysates and multiple, diverse proteins (such as multianalyte pathway elucidation) require the development of a rapid, universal protein quantitation method that is compatible with complex samples. Here, we compare infrared (IR)-based protein quantitation using the Direct Detect™ quantitation system to Bradford and bicinchoninic acid (BCA) colorimetric assays and show that IR-based quantitation provides results that match concentrations determined by amino acid analysis, even in the presence of detergent or reducing agent. We also show that, unlike colorimetric assays, the calculated protein concentration obtained from IR-based analysis is unchanged regardless of the time delay between assay and data acquisition.

The most common colorimetric assays for protein quantitation involve protein-copper chelation (BCA and Lowry assays) and dye-binding based detection (Bradford and "660" assays). While these assays are easy to use, major disadvantages include the large variation in the binding efficiency to different proteins, reproducibility, and sensitivity to sample contaminants. Intrinsic protein characteristics that can affect concentration estimates in colorimetric assays include amino acid content, post-translational modifications, and protein secondary and tertiary structure. In one study, colorimetric assays gave results up to 60% different from values derived from amino acid analysis.