Binding Assays - More Than One Way to Achieve your Goals
Overwhelmed with the abundance of assay options? This blog compares the various advantages and disadvantages of different approaches available for binding assays.
As exact protein concentrations are oftentimes the basis for the success of an experiment, it is worth to dig a little deeper into the background of these assays which potentially saves time and work to get to reliable results. The following lines summarize colorimetric and fluorescent protein measurements and hopefully help to decide for an appropriate assay.
Why quantify proteins?
Just as various as the methods to measure protein concentration are the reasons why proteins need to be quantified in a life science laboratory. Proteins are measured to normalize different protein samples for subsequent applications. The samples contain a mixture of proteins and are lysates of cultured cells, cell compartments or tissues. Based on the protein measurement, they are adjusted to a consistent protein concentration and compared by methods such as western blotting or immunoprecipitations. Quantification of proteins becomes also necessary when the yield of a purification or biotechnological production step is determined. In either case, one typically has multiple samples to analyze which led to the establishment of microplate-based protein measurement assays that enable larger sample sizes and lower assay volumes as compared to their cuvette counterparts.
Which methods are available to measure protein concentration?
Numerous assays are available that quantify the protein content of a sample. They differ in their sensitivity, in their assay principle and in the way of detection. Below you can find the most common protein measurement assays explained and sorted by their way of detection.
Direct absorbance measurement at 280 nm
A direct method of measuring protein is to determine the absorbance of a sample at 280 nm. Aromatic amino acid residues such as Tryptophan and Tyrosine absorb UV-light at 280 nm which allows recalculation of the protein content. No further reagents need to be added to the sample, making it possible to reuse the extract after quantification. However, the method requires the exact extinction coefficient for the protein or protein mixture quantified. The extinction coefficient depends on the amount of aromatic residues and is indispensable to calculate the protein concentration from the absorbance value. Get more information in our topic-related application note here.
Colorimetric protein measurement: Bradford
The Bradford protein quantification assay relies on the association of Coomassie-Brilliant Blue R-250 with proteins and the accompanying shift in absorbance from 470 nm to 595 nm. For the protein measurement, the protein-containing sample is mixed with the Bradford reagent and after 5 min the absorbance at 595 nm is recorded. Parallel to unknown samples, a standard curve with defined amounts of protein is measured which establishes the basis to calculate the concentration of your unknowns.
Typically, bovine serum albumin (BSA) is used as the protein standard. However, choosing a standard that more closely reflects the sample protein may improve the accuracy of the protein concentration measurement. For example, if clones are screened with regard to antibody secretion the use of an IgG standard will provide better results than other protein standards.
Colorimetric protein measurement: BCA (bicinchoninic acid assay)
The BCA assay employs the capability of peptide bonds found in proteins to reduce Cu (II) to Cu+. The copper ion is in turn quantified by BCA which chelates Cu+ and then absorbs light at 562 nm. The BCA assay requires parallel acquisition of a standard curve as well.
Colorimetric protein measurement: Lowry assay
Just as the aforementioned BCA assay, the Lowry protein measurement on the reduction capability of proteins. Again Cu (II) sulfate is reduced to Cu+ which is made visible by Folin-Ciocalteu reagent resulting in a blue complex that can be quantified by its absorbance between 500 and 800 nm. Just like for Bradford and BCA assay, a standard curve is measured in parallel and is the basis for calculating the sample protein concentration.
Fluorescent protein measurements
The NanoOrange® protein quantification assay employs a merocyanine dye that drastically increases fluorescence in the presence of detergent-coated proteins. It can be excited by light around in the blue spectrum (485 nm) and emits in the presence of protein in the yellow/orange spectrum of light, which is reflected in the name of this protein measurement assay. The calculation of protein concentration is again based on the measurement of a protein standard curve.
The fluorescent protein measurement kits Quant iT® Protein and Qubit® Protein work very similar as compared to NanoOrange® (all ThermoFisher Scientific). The Qubit® Protein Assay kit, however, gets along with three standards only. The measurement as well as the analysis is performed with a handheld device that detects fluorescence directly in a reaction tube. The fluorescent nature of the Qubit® assay, however, suggests its transfer to the microplate format.
Overview of protein measurement methods
| Assay | Compatible with detergents? | Concentration range | Comments |
| A280 nm | No | 50 – 2000 mg/ml. | High variation Depends on amino-acid composition of the protein Exact extinction coefficients required Quick |
| Bradford (Coomassie) | Up to 0.1 % in the sample | *125 - 1500 µg/ml | Depends on amino-acid composition of the protein Quick |
| BCA (Bicinchoninic Acid) | Yes | *20 – 2000 µg/ml. | Depends on amino-acid composition of the protein Quick |
| Lowry | No | *1 – 1500 µg/ml | Not dependent on amino acid composition Time-consuming |
| NanoOrange | Limited | *10 ng/ml – 10 µg/ml | |
| Qubit Protein Assay | No | *12.5 µg/ml – 5 mg/ml | Sequential measurements in reaction tube with handheld device |
*concentration ranges are taken from data sheets of commercial suppliers
Depending on the detection modes required, the presented protein measurements can be performed on the SPECTROstar Nano, Omega Series of readers, VANTAstar™, CLARIOstar Plus and PHERAstar FSX.
References
- Bradford, M.M. (1976) Anal, Biochem., 72,248-254.0
- Lowry, O.H. et al. (1951) J. Biol. Chem. , 193, 265-275.
- Smith, P.K. et al. (1985) Anal. Biochem . ,150(1), 76-85.
- Jones, L.J. et al. (2003) Biotechniques. 2003 Apr;34(4):850-4, 856, 858.
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