When studying proteins, a toolset that provides accurate and consistent quantification of protein amounts is necessary to support a wide range of applications.
These include measuring purification yield and quality, normalizing sample inputs, quantifying protein levels in experimental conditions, characterizing protein function, and measuring protein biomarkers.
For this application, pre-packaged protein quantification assay kits are available, which contain the essentials for quantifying total protein or specific proteins from biological samples.
Optimized for ease of use and efficiency, these kits contain all the required reagents and detailed instructions, allowing researchers to quickly perform experiments without extensive preparation.
This guide provides an overview of protein quantification assay kits and some helpful considerations when deciding on which kit to choose.
Benefits of protein assay kits for quantification
Conventional methods of protein quantification, come with benefits and drawbacks.
The spectroscopic absorbance measurement of proteins at the 280 nm wavelength is a simple procedure that requires only a UV-Vis spectrophotometer.
However, these measurements are only reliable for highly purified protein samples in an appropriate, non-interfering buffer.
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Non-protein interference, amino acid content, pH, and solubility are confounding factors that can lead to inaccurate results.
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Protein separation by high-performance liquid chromatography (HPLC) equipped with UV-Vis detectors enables more precise measurements.
This method, however, comes at the cost of expensive instrumentation, expertise, and a more complex workflow.
Protein assay and quantification kits offer the sensitivity and resolution ideal for analytical measurements while also retaining the flexibility and simplicity of spectrophotometer-based methods.
Many assay kits can detect very low protein concentrations in the nanogram to microgram range, which is essential for samples containing dilute or low-abundance proteins.
They can also operate over a broad dynamic range to accurately measure a wide range of protein concentrations.
This is useful when dealing with multiple samples in which protein levels can vary significantly.
The detection chemistry of some kits can also accommodate complex samples with interfering substances, such as salts, detergents, and cellular debris.
This flexibility allows the quantification of different samples such as cell lysates, tissue extracts, serum, or plasma, with minimal preparative steps.
Containing complete and optimized sets of reagents, protein assay kits enable researchers to quickly obtain experimental results.
Many kits are also compatible with high-throughput screening formats, such as 96-well or 384-well plates, which can improve throughput and efficiency in large-scale studies.
Common assays for total protein
Bradford Assay -
The well-known Bradford protein assay uses the Coomassie Brilliant Blue G-250 dye, which strongly binds proteins to form a blue protein-dye complex.
This colorimetric reaction corresponds with an increase in absorption at 595 nm, proportional to the concentration of proteins in the sample.
Bradford assay kits generally have a protein detection range of 125 – 1,500 µg/mL.
It is a straightforward assay that involves only the addition of the Coomassie dye solution with an incubation time of as little as 2 minutes.
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It is resistant to interference from small quantities of salts, carbohydrates and reducing agents, but the presence of detergents such as SDS and Triton X-100 can interfere with the color of the assay.
Overall, the Bradford assay is a fast and reliable method for routine protein quantification when detergents are not significantly present in the sample.
Biuret Assay -
The Biuret assay is one of the earliest methods of protein quantification, dating as early as 1833.
In this assay, the presence of at least two peptide bonds in an alkaline solution reacts with copper ions to form a purple complex.
Biuret assay kits utilize the biuret reagent to react with proteins (of at least 3 amino acids) for colorimetric detection at the 540 nm wavelength.
Many assay kits feature a detection range of 0.58 - 100 g/L.
BCA Assay -
Defined by its use of the bicinchoninic acid (BCA) reagent, the BCA assay utilizes the conversion of Cu2+ to Cu+ in alkaline conditions, resembling the Lowry and Biuret assays.
To detect the formation of Cu+, BCA can be added in a one-step procedure due to its stability under alkali conditions.4
The result is a purple colorimetric reaction with an absorbance maximum at 562 nm.
BCA assay kits generally have a detection range of 20 – 2000 µg/mL, while some microscale kits can accommodate as low as 4 to 40 µg/ml.
It is resistant to reasonable concentrations of common detergents (eg. SDS, Triton X-100, Tween) and denaturing agents (eg. urea and guanidinium chloride).
However, it is susceptible to metal chelators (eg. EDTA and EGTA), reducing agents (DTT, mercaptoethanol), and lipids.
This assay can be an ideal option when quantifying proteins directly extracted from tissue and cell lysates.
Assays for specific proteins
In addition to measuring total protein content, biochemical assays are available for detecting specific proteins or protein modifications.
These assays are valuable for the rapid quantification of biomarkers or for monitoring biological processes.
Some common examples are outlined below.
Protein Carbonyl Assay -
Protein carbonyls can arise from the oxidation of protein side chains by reactive oxygen species.
Protein carbonylation is thus an often-used marker for oxidative stress.
Protein carbonyl assays often involve the reaction of 2,4-dinitrophenylhydrazine (DNPH) with protein carbonyl groups to form a stable hydrazone product.
Hydrazone formation can be measured by spectroscopic absorbance or detected by specific antibodies.
Albumin Assay -
Albumin assays are designed for the specific quantification of albumins, a family of ligand-binding proteins and a major component of blood.
Albumin assay kits often utilize albumin-selective probes or dyes (such as bromocresol green) that generate chromogenic or fluorescence signals proportional to the concentration of albumin proteins in the sample.
Some assays also use specific antibodies with enzyme labels for detection.
Hemoglobin Assay -
Hemoglobin is a protein complex found in red blood cells and is often used as a diagnostic biomarker.
Hemoglobin assays generally involve the conversion of hemoglobin into a stable colored complex using reactants such as O-tolidine.
The increase in spectroscopic absorbance of the reaction is proportional to the concentration of hemoglobin.
Hemoglobin assay kits typically can detect at concentrations as low as 10 µg/mL.
Considerations for protein assays
Be aware of confounding agents.
As mentioned above, certain protein assays are susceptible to interfering agents.
Users should be fully aware of the chemical constitution of their protein samples.
Are there detergents, lipids, reducing agents, or chelators—and if so—at what concentrations?
The upstream procedures in a workflow will influence sample composition (eg. detergent-based cell lysis, SDS-PAGE gel extractions).
Refer to the assay protocol in advance to learn more about the compatiblity of the protein sample and along with other dissolved components.
Review the assay protocol.
The assay protocol, sometimes called the product manual, is a valuable resource that offers key information for prospective users.
Such details include any additional tools or instrumentation not supplied by the kit, the linear range at which protein concentrations can be reliably measured, and the format of supplied protein standards (if included).
While protein assays generally share common workflows, requirements and limitations, some variations can exist between different kits.
Some kits may feature improved assay reagents or specialized instructions for high-throughput or micro-scale assay formats.
Others may also include reagents for cleaning up samples prior to the assay to remove interfering components and improve assay reliability.
References
1. DePalma, A. Quantifying Protein Expression. Biocompare. 2021 Aug 12 [cited 2024 Sep]. Available from: www.biocompare.com/Editorial-Articles/578072-Quantifying-Protein-Expression/
2. Long, MS. Frontiers in Protein Quantitation. Biocompare. 2024 Jun 13 [cited 2024 Sep]. Available from: www.biocompare.com/Editorial-Articles/613186-Frontiers-in-Protein-Quantitation/
3. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248-254. doi:10.1006/abio.1976.9999
4. Walker JM. The bicinchoninic acid (BCA) assay for protein quantitation. Methods Mol Biol. 1994;32:5-8. doi:10.1385/0-89603-268-X:5
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