Protein concentration quantitation is an integral part of any laboratory workflow involving protein extraction, purification, labeling or analysis. Pierce Protein Assays provide a wide range of options for accurate protein concentration determination based on assay time, sensitivity, compatibility, standard curve linearity, and protein-to-protein variation. Although this article uses Pierce Protein Assay products as examples, the principles and chemistries discussed apply generally to most available colorimetric or fluorometric protein assay techniques.
Protein quantitation is often necessary before processing protein samples for isolation, separation and analysis by chromatographic, electrophoretic and immunochemical techniques. Depending on the accuracy required and the amount and purity of the protein available, different methods are appropriate for determining protein concentration.
The simplest and most direct assay method for protein concentration determination in solution is to measure the absorbance at 280 nm (UV range). Amino acids containing aromatic side chains (i.e., tyrosine, tryptophan and phenylalanine) exhibit strong UV-light absorption. Proteins and peptides absorb UV-light in proportion to their aromatic amino acid content and total concentration. Another method, traditionally used in amino acid analysis by HPLC, is to label all primary amines (i.e., N-terminus and side-chain of lysine residues) with a colored or fluorescent dye such as ninhydrin or o-phthalaldehyde (OPA). Direct UV-light absorbance and HPLC-reagent approaches have particular disadvantages that make these methods impractical for use with typical protein samples in proteomics workflows. UV absorption method is not ideal for protein mixtures as different proteins have differing aromatic amino acid content- varying the absorption characteristics. In addition, any non-protein content that absorbs UV light will interfere with measurements.
To overcome these disadvantages, several colorimetric and fluorescent, reagent-based protein assay techniques have been developed that are used by nearly every laboratory involved in protein research. Protein samples are added to the reagent, producing a color change or increased fluorescence in proportion to the amount added. Protein concentration is determined by reference to a standard curve consisting of known concentrations of a purified reference protein. These protein assay techniques can be divided into two groups based on the type of chemistry involved.
Table 1. Types, advantages, disadvantages and examples of protein assay methods.
|Method||Advantages||Disadvantages||Example assay reagents|
|Biuret methods: Protein-copper chelation and secondary detection of reduced copper||BCA Assays|
|Colorimetric dye based methods: Protein-dye binding and direct detection of the color change||Bradford (Coomassie)|
|Fluorescent dye methods: Protein-dye binding and direct detection of increase in fluorescence associated with the bound dye||EZQ fluorescent assay|
Qubit Protein Assay
Selecting a protein assay
No one reagent can be considered to be the ideal or best protein assay method. Each method has its advantages and disadvantages. The choice among available protein assays is usually based on the compatibility of the protein assay method with the samples. Additionally, one must consider potential interfering substances included in samples that may affect certain assay methods, as well as the accuracy, reproducibility and incubation time desired. Therefore, successful use of protein assays involves selecting the method that is most compatible with the samples to be analyzed, choosing an appropriate assay standard, and understanding and controlling the particular assumptions and limitations that remain.
The objective is to select a method that requires the least manipulation or pre-treatment of the samples to accommodate substances that interfere with the assay. Each method has its particular advantages and disadvantages. Because no one reagent can be considered the ideal or best protein assay method for all circumstances, most researchers have more than one type of protein assay available in their laboratories.
Important criteria for choosing an assay include:
- Compatibility with the sample type and components
- Assay range and required sample volume
- Protein-to-protein uniformity (see below)
- Speed and convenience for the number of samples to be tested
- Availability of spectrophotometer or plate reader necessary to measure the color produced (absorbance) by the assay
Some common substances that potentially interfere with protein assay methods are reducing agents (e.i. DTT) and detergents (i.e. Triton X-100). In general, samples containing reducing agents or copper-chelating agents are preferentially analyzed with Coomassie dye–based assays (Bradford method). This is because Coomassie dye–based assays, such as the Pierce Coomassie (Bradford) and Pierce Coomassie Plus (Bradford) assays, are compatible with reducing agents and do not require copper-protein binding reactions. For those samples that contain detergents, copper-based protein assays such as the Pierce Rapid Gold BCA assay are the better choice as they are not inhibited by low to moderate amounts of detergent.
In addition to sample compatibility, protein assays are also commonly grouped by the range of protein concentrations they can detect. For samples in which protein concentration is expected to be low (<20 μg/mL), it may be necessary to use an assay’s alternate microplate protocol or use a specialized assay, such as the Pierce Micro BCA Protein Assay, which is specifically designed for dilute samples. If the total protein concentration in the samples is high (>2,000 μg/mL), sample dilution can often be used to overcome any problems with known interfering substances. Sometimes the sample contains substances that make it incompatible with any of the protein assay methods. The preferred method of dealing with these types of interfering substances is to simply remove them.
The Pierce Rapid Gold BCA Protein Assay and Coomassie (Bradford) Protein Assay complement one another and provide the two basic methods for accommodating most samples. The various accessory reagents and alternative versions of these two assays accommodate many other particular sample needs.
Selecting a protein standard
Because proteins differ in their amino acid compositions, each one responds somewhat differently in each type of protein assay. Therefore, the best choice for a reference standard is a purified, known concentration of the most abundant protein in the samples. This is usually not possible to achieve, and it is seldom convenient or necessary. In many cases, the goal is merely to estimate the total protein concentration, and slight protein-to-protein variability is acceptable.
If a highly purified version of the protein of interest is not available or it is too expensive to use as the standard, the alternative is to choose a protein that will produce a very similar color response curve in the selected protein assay method and is readily available to any laboratory at any time. Generally, bovine serum albumin (BSA) works well for a protein standard because it is widely available in high purity and relatively inexpensive. Alternatively, bovine gamma globulin (BGG) is a good standard when determining the concentration of antibodies because BGG produces a color response curve that is very similar to that of immunoglobulin G (IgG).
For greatest accuracy in estimating total protein concentration in unknown samples, it is essential to include a standard curve each time the assay is performed. This is particularly true for the protein assay methods that produce non-linear standard curves. Deciding on the number of standards and replicates used to define the standard curve depends upon the degree of non-linearity in the standard curve and the degree of accuracy required. In general, fewer points are needed to construct a standard curve if the color response is linear. Typically, standard curves are constructed using at least two replicates for each point on the curve.
Sample preparation for protein assays
Before a sample is analyzed for total protein content, it must be solubilized, usually in a buffered aqueous solution. Additional precautions are often taken to inhibit microbial growth or to avoid casual contamination of the sample by foreign debris such as dust, hair, skin or body oils.
Depending on the source material that the procedures involved before performing the protein assay, the sample will contain a variety of non-protein components. Awareness of these components is critical for choosing an appropriate assay method and evaluating the cause of anomalous results. For example, tissues and cells are usually lysed with buffers containing surfactants (detergents), biocides (antimicrobial agents) and protease inhibitors. Different salts, denaturants, reducing agents and chaotropes may also be included. After filtration or centrifugation to remove the cellular debris, typical samples will still include nucleic acids, lipids and other non-protein compounds.
Every type of protein assay is adversely affected by substances of one sort or another. Components of a protein solution are considered interfering substances in a protein assay if they artificially suppress the response, enhance the response, or cause elevated background by an arbitrarily chosen degree (e.g., 10% compared to control).
Inaccuracy resulting from a small amount of interfering substance can be eliminated by preparing the protein standard in the same buffer as the protein being assayed. For higher, incompatible levels of interfering substances, other strategies are necessary:
- Choose a different protein assay method or a version of the same assay method that includes components to overcome the interference.
- Dialyze or desalt the sample to remove interfering substances that are small (i.e., less than 1000 daltons), such as reducing agents.
- Precipitate the protein in TCA or other appropriate reagent, remove the solution containing the interfering component, and then re-dissolve the protein for analysis. This illustration provides an overview of how protein dialysis methods are used to remove substances that may contaminate protein samples and interfere with downstream applications.
Figure 1. The schematic here shows how a dialysis cassette can be used for protein cleanup. 3 mL of 1 mg/mL IgG in 0.1 M Tris buffer, pH 7 inside a dialysis cassette is placed in 1,000 mL of 100 mM PBS, with a pH of 7.6. The old dialysate is discarded and replaced with 1,000 mL of 100 mM PBS, with a pH of 7.6. IgG is too large to enter the pores in the membrane; therefore, the amount of IgG inside the cassette remains constant. The Tris buffer concentration drops below 0.01 M inside the cassette as the Tris buffer diffuses out and the PBS buffer diffuses in. Again, the old dialysate is discarded and replaced with 1,000 mL of 100 mM PBS, with a pH of 7.6. The IgG inside of the cassette remains constant. The Tris buffer inside of the cassette drops to near undetectable levels. The buffer inside the cassette is 100 mM PBS, with a pH of 7.6.
Each protein in a sample responds uniquely in a given protein assay. Such protein-to-protein variation refers to differences in the amount of color (absorbance) obtained when the same mass of various proteins is assayed concurrently by the same method. These differences in color response relate to differences in amino acid sequence, isoelectric point (pI), secondary structure and the presence of certain side chains or prosthetic groups.
Depending on the sample type and purpose for performing an assay, protein-to-protein variation is an important consideration in selecting a protein assay method and in selecting an appropriate assay standard (e.g., BSA vs. BGG). Protein assay methods based on similar chemistry have similar protein-to-protein variation.
Figure 2. Standard curves. Example standard curves using purified BSA and BGG with Pierce BCA Protein Assay Kit illustrating the differences in color intensities produced from the two different proteins.
Protein-protein variation of protein assays
Protein assays differ in their chemical basis for detecting protein-specific functional groups. Some assay methods detect peptide bonds, but no assay does this exclusively. Instead, each protein assay detects one or several different particular amino acids with greater sensitivity than others. Consequently, proteins with different amino acid compositions produce color at different rates or intensities in any given protein assay.
The following table compares the protein-to-protein variability in color response of several Thermo Scientific Pierce Protein Assays. These data serve as a general guideline for evaluating response differences among protein samples. However, because the comparisons were made using one protein concentration and buffer, they should not be used as exact calibration factors.
This variability information is helpful for choosing a protein standard. For example, when the sample to be assayed is a purified antibody, bovine gamma globulin (BGG, protein #5) will be a more accurate standard than bovine serum albumin (BSA, protein #1). These data also indicate the importance of specifying which assay standard was used when reporting protein assay results.
For each of the protein assays presented here, 14 proteins were assayed using the standard test tube protocol. The net (blank corrected) average absorbance for each protein was calculated. The net absorbance for each protein is expressed as a ratio to the net absorbance for BSA (e.g., a ratio of 0.80 means that the protein produces 80% of the color obtained for an equivalent mass of BSA). All protein concentrations were at 1000 µg/mL, except with the Micro BCA Assay which were at a concentration of 10 µg/milk.
Table 2. Overview of Protein Assays.
|Relative uniformity||High||High||High||Medium||Low (Note 2)||Low|
|Coefficient of variation||14.7%||11.4%||11.9%||28.8%||38.2%||37%|
|1. Albumin, bovine serum||1.00||1.00||1.00||1.00||1.00||1.00|
|2. Aldolase, rabbit muscle||0.85||0.80||0.94||0.74||0.76||0.83|
|4. Cytochrome C, horse heart||0.83||1.11||0.94||1.03||1.07||1.22|
|5. Gamma globulin, bovine||1.11||0.95||1.14||0.58||0.56||0.51|
|6. IgG, bovine||1.21||1.12||1.29||0.63||0.58||—|
|7. IgG, human||1.09||1.03||1.13||0.66||0.63||0.57|
|8. IgG, mouse||1.18||1.23||1.20||0.62||0.59||0.48|
|9. IgG, rabbit||1.12||1.12||1.19||0.43||0.37||0.38|
|10. IgG, sheep||1.17||1.14||1.28||0.57||0.53||—|
|11. Insulin, bovine pancreas||1.08||1.22||1.12||0.67||0.60||0.81|
|12. Myoglobin, horse heart||0.74||0.92||0.90||1.15||1.19||1.18|
|14. Transferrin, human||0.89||0.98||0.92||0.90||0.84||0.8|
|17. Trypsin inhibitor, soybean||—||—||—||—||—||0.38|
|Protein-to-protein variation of Thermo Scientific Pierce Protein Assays Notes:|
1. The BCA - Reducing Agent Compatible (BCA-RAC) Assay also produced a low coefficient of variation.
2. The Bio-Rad Bradford Protein Assay tested with the same proteins as our Coomassie (Bradford) Assay produced a very high coefficient of variation (46%), corresponding to very low relative uniformity.
Calculations and data analysis
With most protein assays, sample protein concentrations are determined by comparing their assay responses to that of a dilution-series of standards whose concentrations are known. Protein samples and standards are processed in the same manner by mixing them with assay reagent and using a spectrophotometer to measure the absorbances. The responses of the standards are used to plot or calculate a standard curve. Absorbance values of unknown samples are then interpolated onto the plot or formula for the standard curve to determine their concentrations.
Obviously, the most accurate results are possible only when unknown and standard samples are treated identically. This includes assaying them at the same time and in the same buffer conditions, if possible. Because different pipetting steps are involved, replicates are necessary if one wishes to calculate statistics (e.g., standard deviation, coefficient of variation) to account for random error.
Figure 3. Comparison of point-to-point and linear-fit standard curves. Interpolation and calculation for a test sample having absorbance 0.6 results in significantly different protein concentration values. In this case, the point-to-point method clearly provides a more accurate reference line for calculating the test sample.
Although most modern spectrophotometers and plate readers have built-in software programs for protein assay data analysis, several factors are frequently misunderstood by technicians. Taking a few minutes to study and correctly apply the principals involved in these calculations can greatly enhance one's ability to design assays that yield the most accurate results possible (see the related Tech Tips and links).
- Download: Determine wavelengths for measuring protein assays
- Download: How to use a standard curve
Peptide quantitation assays
For workflows utilizing proteomics using mass spectrometry, it is important to measure peptide concentration following protein digestion, enrichment, and/or C18 clean-up steps in order to normalize sample-to-sample variation. In particular, for experiments utilizing isobaric labeling, it is critical to ensure that equal amounts of sample are labeled before mixing in order to have accurate results.
Similar to protein assay methods, various options are available for determining peptide concentration. Historically, UV-Vis (A280) or colorimetric, reagent-based protein assay techniques have been employed to measure peptide concentrations. Both BCA and micro-BCA assays are frequently used. Although these strategies work well for protein samples, these reagents are not designed for accurately detecting peptides. Alternatively, quantitative peptide assays— in either a colorimetric or fluorometric format—are available to specifically quantitate peptide mixtures. When deciding to use a colorimetric or fluorometric microplate assay format for quantitative peptide assays these important criteria must be considered:
- Compatibility with the sample type, components, and workflows
- Assay range and required sample volume
- Speed and convenience for the number of samples to be tested
- Availability of the spectrophotometer or fluorometer needed to measure the output of the assay
This representative data compares results obtain using colorimetric and fluorometric assays.
Figure 4. Quantitation comparison between colorimetric and fluorometric peptide assays. Tryptic peptide digests were prepared from twelve cell lines. Peptide digest concentrations were determined using the Thermo Scientific Pierce Quantitative Colorimetric Peptide Assay and the Pierce Quantitative Fluorometric Peptide Assay Kits according to instructions. Each sample was assayed in triplicate, and the concentration of each digest was calculated with standard curve generated using the Protein Digest Assay Standard.
- Bradford, MM. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry. 72, 248-254.
- Smith, P.K, Krohn R.I., Hermanson G.T., et al. (1987) Measurement of protein using bicinchoninic acid. Analytical Biochemistry. 150, 76-85.
- Krohn, R.I. (2002). The Colorimetric Detection and Quantitation of Total Protein, Current Protocols in Cell Biology, A.3H.1-A.3H.28, John Wiley & Sons, Inc.
- Krohn, R.I. (2001). The Colorimetric Determination of Total Protein, Current Protocols in Food Analytical Chemistry , B1.1.1-B1.1.27, John Wiley & Sons, Inc.
- Lowry, O.H., Rosebrough, N.J., Farr, A.L., et al. (1951) Protein measurement with folin phenol reagent. Journal of Biological Chemistry. 193, 265-75.
- Legler G, Müller-Platz CM, Mentges-Hettkamp M, et al. (1985) On the chemical basis of the Lowry protein determination. Analytical Biochemistry. 150, 278-87.
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