Overview of Protein Assays Methods
Protein quantitation is an integral part of any laboratory workflow involving protein extraction, purification labeling or analysis. Cell lysates are assayed to verify success of the lysis step and to normalize multiple samples for storage or side-by-side comparison. Proteins obtained from a purification procedure are assayed to determine yield. Purified proteins that will be labeled with biotin or conjugated to reporter enzymes are typically assayed to ensure that the labeling reaction is prepared with appropriate stoichiometry. Given the wide range of chemicals that may be present in different kinds of samples, it is amazing that there exist protein assay reagents that are capable of reliably and specifically measuring the protein component.
Thermo Scientific™ Pierce™ Protein Assays provide exceptional accuracy, compatibility and broad applicability that enable most laboratory protein samples to be determined with ease. Although this article uses Pierce Protein Assay products as examples, the principles and chemistries discussed apply generally to most available colorimetric 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 proteins in solution is to measure the absorbance at 280nm (UV range). Amino acids containing aromatic side chains (i.e., tyrosine, tryptophan and phenylalanine) exhibit strong UV-light absorption. Consequently, 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-phthaldialdehyde (OPA). Direct UV-light absorbance and HPLC-reagent approaches have particular disadvantages that make them impractical for use with typical protein samples in proteomics workflows.
Instead, several colorimetric, reagent-based protein assay techniques have been developed that are used by nearly every laboratory involved in protein research. Protein is added to the reagent, producing a color change in proportion to the amount added. Proteins 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.
|Type: Assay based on||Example Thermo Scientific Pierce Protein Assays|
|Protein-copper chelation and secondary detection of the reduced copper||
|Protein-dye binding and direct detection of the color change associated with the bound dye||Coomassie (Bradford)
- Tech Tip #6: Extinction coefficients and protein concentration
The 40-page handbook reviews the principle of four major protein assay chemistries and contains an updated substance compatibility list. In addition, reaction schemes, protein-to-protein variation data, protocol schematics and a quick technical summary table accompany each assay methodology. The table includes the working range of the assay, characteristics and advantages, applications, disadvantages, and interfering substances. Useful references are provided with each assay.
Unfortunately, no protein assay method exists that is either perfectly specific to proteins (i.e., not affected by any nonprotein components) or uniformly sensitive to all protein types (i.e., not affected by differences in protein composition). 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.
When it is necessary to determine total protein concentration of a sample, one must first select an appropriate protein assay method based upon its compatibility with the samples type. 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
The Thermo Scientific™ Pierce™ BCA Protein Assay and Thermo Scientific™ Pierce™ 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.
- Tech Tip #68: Protein assay compatibility table
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.
- Tech Tip #57: How to use a protein assay standard curve
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 nonprotein components. Awareness of these components is critical for choosing an appropriate assay method and evaluating the cause of anomolous 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 redissolve the protein for analysis. Our Thermo Scientific™ Compat-Able™ products (see side bar) include reagents for this purpose.
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.
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.
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 principles 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).
For Research Use Only. Not for use in diagnostic procedures.