Comparison of the Quant-iT™ Protein Assay and Other Conventional Assays

Comparison of the Quant-iT™ Protein Assay and Other Conventional Assays

The detection and quantitation of proteins are vital to many biological studies, due to the ubiquitous and fundamental roles of proteins in biological processes as well as the commercial importance of proteins in the biotech and pharmaceutical industries.

There are currently several absorbance- and fluorescence-based protein assays in common use, each with its own shortcomings, such as protein-to-protein variability, contaminant interference, time requirements, accuracy, sensitivity, and the need for caustic or hazardous reagents. We compared four common protein assays for protein-to-protein variation, accuracy, precision, and sensitivity. The Quant-iT™ Protein Assay for the Qubit® Fluorometer compared favorably by providing low protein variability, rapid quantitation, accuracy, precision, and high sensitivity.

NOTE: Since this article was published, we have launched the next generation instrument, the Qubit® 2.0 Fluorometer which quantitates DNA, RNA, and protein with unprecedented accuracy, sensitivity, and simplicity.

The Qubit™ Quantitation Platform: Fast and Easy to Use

The Qubit™ Quantitation Platform is the combination of a user-friendly fluorometer with highly sensitive fluorescence-based quantitation assays. The Qubit® Fluorometer is a small, economical instrument designed to work seamlessly with Quant-iT™ Assay Kits for routine protein, DNA, and RNA quantitation (Figure 1). All settings and calculations are performed for you. The system is simple, fast, and easy to use, yet consistently produces accurate results, so you can be confident moving forward with subsequent applications. Each Quant-iT™ Assay Kit is highly specific for a single analyte, and all are more sensitive than absorbance-based measurements. Only small sample volumes of 1–20 μL are required, and all assay reagents are stored at room temperature, eliminating the need to thaw reagents.

Workflow for Quant-iT™ assays with Qubit® Fluorometer

Figure 1. Workflow for the Quant-iT™ assays using the Qubit® Fluorometer.

Quant-iT™ Protein Assay: Accurate, Precise, and Sensitive

We compared 3 assays to the Quant-iT™ Protein Assay for the Qubit® Fluorometer (Figure 2): the Bio-Rad® Quick Start™ Bradford Protein Assay, the Pierce® BCA Protein Assay, and the Pierce® Modified Lowry Protein Assay. The Bradford method (using Coomassie Brilliant Blue) [1] exhibited very high protein-to-protein variability (Figure 2B). Spectrophotometric assays such as those using BCA (bicinchoninic acid) [2] require carefully timed steps, are not compatible with reducing agents, and often yield high estimates of protein, as observed for lysozyme in our study (Figure 2C). The Lowry method [3] (Figure 2D) employs a lengthy, multistep procedure and is incompatible with detergents, carbohydrates, and reducing agents. The Quant-iT™ Protein Assay showed low protein-to-protein variation, good accuracy and precision, and sensitivity down to 0.025 mg/mL in the stock sample (Figure 2A).

The Quant-iT™ Protein Assay is insensitive to many common contaminants, including reducing agents, nucleic acids, and free amino acids. However, detergents, such as SDS (final concentration >0.01%), Tween® 20, and Triton® X-100, are not recommended. The assay has an optimal range of 1.25 to 25 µg/mL (0.25–5 µg) in the assay tube (initial stock concentrations, 12.5–5 mg/mL) and is provided in a simple kit format that allows easy and rapid use. Learn more about the Qubit™ Quantitation Platform.

Figure 2. The Quant-iT™ Protein Assay with the Qubit® Fluorometer produces less protein-to-protein variation and higher accuracy, precision, and sensitivity than 3 other common protein assays. (A–D) The same lot of each protein was used in all assays, and assays were carried out in triplicate following the manufacturers’ protocols. Data are graphed to show protein-to-protein variation throughout the protein concentration range tested. The inset in (A) is a magnification of the low end of the protein range, to show the sensitivity of the Quant-iT™ Protein Assay used with the Qubit® Fluorometer.