Protein Assays and Quantitation Support—Getting Started

We offer several types of protein assays including the: BCA Assay, BCA-RAC (Reducing Agent Compatible) Assay, Micro BCA Assay, 660 nm Protein Assay, Pierce Bradford Plus Protein Assay Kit, Pierce Bradford Protein Assay Kit, Modified Lowry Assay, colorimetric and fluorometric Peptide Assays, CBQCA kit, EZQ kit, Quant-iT kits, NanoOrange, and the Qubit kits.

Typically, peptides need to be around 2 or 3 kDa (depending on the protein assay and the exact peptide composition) to be measured using a protein assay. For peptides, we offer two quantitative assays, the Quantitative Fluorometric Peptide Assay (Cat. No. 23290) and the Quantitative Colorimetric Peptide Assay (Cat. No. 23275). 

We offer several “specialty” protein assays including: Protease assay kits (Cat. Nos. 23263 and 23266), Glycoprotein Carbohydrate Estimation Kit (Cat. No. 23260), Phosphoprotein Phosphate Estimation Kit (Cat. No. 23270), Quantitative Peroxide assay kits (Cat. Nos. 23280 and 23285), and Easy-Titer™ Assay kits for IgG and IgM.

Unfortunately, no protein assay method exists that isn’t affected by any non-protein component or uniformly sensitive to all protein types. One must select an appropriate assay method based on compatibility with the sample type or one that requires the least manipulation of the sample to accommodate the assay. Most researchers will have more than one type of assay available in their laboratories. 

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. 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.

There are several criteria that should be considered, including compatibility with the sample type and components, assay range and required sample volume, protein-to-protein uniformity, speed and convenience for the number of samples to be tested, and the availability of spectrophotometer or plate reader necessary to measure the color produced (absorbance) by the assay.

Before the sample is analyzed, it must be solubilized; usually, in a buffered aqueous solution. Depending on the source material and 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. 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). Additional components can include reducing agents, chelators, crowding agents, and protease inhibitors. 

Each protein in a sample responds uniquely in a given protein assay, and this protein-to-protein variation is observed as 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.

The Glycoprotein Carbohydrate Estimation Kit (Cat. No.23260) enables the amount of protein glycosylation to be measured as the percent of total purified protein mass.

The Phosphoprotein Phosphate Estimation Kit (Cat. No. 23270) enables classification and identification of proteins as phosphorylated serine and threonine, as well as semi-quantitative assessment of the phosphorylation level.

The maximum absorbance of the Dilution-Free Rapid Gold BCA complex is 480 nm (the complex produces an orange-gold colored reaction product). Although the color may be measured at any wavelength between 460-500 nm, the standard curve slope and overall assay sensitivity will be slightly reduced (<10%).

For peptide sample concentration measurements, we recommend using either the Pierce Quantitative Fluorometric Peptide Assay (Cat. No. 23290) or the Pierce Quantitative Colorimetric Peptide Assay (Cat. No. 23275).

Certain substances are known to interfere with the BCA assay, including those with reducing potential, chelating agents, and strong acids or bases. For the assay compatibility of various substances, please see Table 3 in the Dilution-Free Rapid Gold BCA Protein Assay manual.

Most colorimetric protein assay methods can be divided into two groups based on the type of chemistry involved: those involving protein-copper chelation with secondary detection of the reduced copper and those based on protein-dye binding with direct detection of the color change associated with the bound dye.

Copper-based protein assays, including the BCA and Lowry methods, depend on the well-known "biuret reaction", whereby peptides containing three or more amino acid residues form a colored chelate complex with cupric ions (Cu2+) in an alkaline environment containing sodium potassium tartrate.

Pierce Bradford Protein Assay Kit and Pierce Bradford Plus Protein Assay Kit are variations on the use of Coomassie G-250 dye as a colorimetric reagent for the detection and quantitation of total protein first reported by Bradford in 1976. The Thermo Scientific 660 nm Protein Assay is a dye-based reagent that offers the same convenience as Coomassie-based assays while overcoming several of their disadvantages. In particular, the 660 nm Assay is compatible with most detergents and produces a more linear response curve (the detailed assay chemistry is proprietary). Our fluorometric protein assays are also based on dye binding chemistries.

• Identically assayed samples are directly comparable: Sample assay responses are directly comparable to each other if they are processed in exactly the same manner. Variation in amount of protein is the only possible cause for differences in final absorbance (color intensity) if the samples are dissolved in the same buffer, the same lot and stock of assay reagent is used, all samples are mixed and incubated at the same time and temperature, and no pipetting errors were introduced. 
• Units in equals the units out: The unit of measure used to express the standards is by definition the same unit of measure associated with the calculated value for the unknown sample (i.e., final results for unknown samples will be expressed in the same unit of measure as was used for the standards).

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. 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 as 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).

Protein concentrations are generally determined and reported with reference to standards of a common protein, such as bovine serum albumin (BSA). If precise quantitation of an unknown protein is required, it is advisable to select a protein standard that is similar in quality to the unknown; for example, a bovine gamma globulin (BGG) standard may be used when assaying immunoglobulin samples.

Protein standards should preferably be diluted using the same diluent as the sample(s). Sample assay responses are directly comparable to each other if they are processed in exactly the same manner. Variance in protein quantity is the only possible cause for differences in final absorbance (color intensity) if samples are dissolved in the same buffer and the same stock solution of assay reagent is used for all samples. 

However, if only a “rough” estimate of protein concentration is needed, a blank-only correction can be used. In this case, a blank is prepared in the diluent of the sample to correct for its raw absorbance. The concentration of the sample is then determined from a standard curve obtained from a series of dilutions of the protein of known concentration prepared in water or saline solution. 

Yes, we recommend storing at -20 degrees C and they will likely be good for 2-3 months, or about 2 freeze/thaws. 

The unit of measure used to express the standards is by definition the same unit of measure associated with the calculated value for the unknown sample (i.e., final results for unknown samples will be expressed in the same unit of measure as was used for the standards). For example, if the standards are expressed as micrograms per milliliter, then the value for the unknown sample, which is determined by comparison to the standard curve, is also expressed as micrograms per milliliter.

Simply multiply the calculated concentration of the diluted sample by the dilution factor. For example: A protein sample is known to be approximately 5 mg/mL. This is too concentrated to be assayed by the Pierce Bradford Plus Protein Assay Kit, whose assay range in the standard microplate protocol is 100-1500 µg/mL. However, you could dilute it 5-fold in buffer (i.e., 1 part sample plus 4 parts buffer) and then use that diluted sample as the test sample in the protein assay. If the test sample produces the same absorbance as the 1000 µg/mL standard sample, then you can conclude that the test (5-fold diluted) sample is 1000 µg/mL, and therefore the original (undiluted) sample is 5 × 1000 µg/mL = 5000 µg/mL = 5 mg/mL.

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. 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. 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 you wish to calculate statistics (e.g., standard deviation, coefficient of variation) to account for random error.

Most modern plate readers and spectrophotometers have associated software that automatically plots a linear or curvilinear regression line through the standard points, interpolates the test samples on that regression line, and reports the calculated value. However, there are different methods for making the calculations “by hand”. You can find a detailed explanation and example in our Tech Tip. 

Enter the concentration values for the standards in Column A and their corresponding absorbance data in Column B. Highlight both columns and from the Insert menu select Chart and XY (Scatter). Click on the resulting graph and select Add Trendline from the Chart menu. While viewing the graph next to the open Format Trendline window, choose Polynomial and set the Order to 2, 3 or 4 until the best-fit appears. Check the box near the bottom called Display Equation on Chart; then close the Format Trendline window. Use the resulting equation to determine protein concentration (y) of an unknown sample by inserting the sample’s absorbance value (x). 

No. Contrary to what many people assume, it is neither necessary nor even helpful to know the actual amount (e.g., micrograms) of protein applied to each well or cuvette of the assay. The amount of protein per well is almost certainly not the value of interest; instead, one usually wants to know the protein concentration of the original test sample.

No. It is neither necessary nor helpful to know the protein concentration as it exists when diluted in assay reagent. The protein concentration when diluted by assay reagent is almost certainly not the value of interest; instead, one wants to know the protein concentration of the original test sample.

One situation in which the dilution factor is important to consider is when the original sample has been pre-diluted relative to the standard sample. Suppose the original protein sample is actually known to be approximately 5 mg/mL. This is too concentrated to be assayed by the Pierce Bradford Plus Protein Assay Kit, for example, whose assay range in the standard microplate protocol is 100-1500 µg/mL. However, you could dilute it 5-fold in buffer (i.e., 1 part sample plus 4 parts buffer) and then use that diluted sample as the test sample in the protein assay. If the test sample produces the same absorbance as the 1000 µg/mL standard sample, then you can conclude that the test (5-fold diluted) sample is 1000 µg/mL, and therefore the original (undiluted) sample is 5 x 1000 µg/mL = 5000 µg/mL = 5 mg/mL.

Several factors affect protein assay accuracy and precision:

Replicates: The only way to evaluate the extent of random error is to include replicates of each standard and test sample. Because all test samples are evaluated by comparison to the standard curve, it is especially important to run the standards in triplicate. The standard deviation (SD) and coefficient of variation (CV) can then be calculated, providing a degree of confidence in your pipetting precision. If replicates are used, curve-fitting is done on the average value (minus obvious outliers).

Blank correction: It is common practice to subtract the absorbance of the zero assay standard(s) from the all other sample absorbance values. However, if replicate zero-assay standards will be used to calculate error statistics, then another independent value may be required for blank-correction. If the standards were prepared in a buffer to match that of the test samples, and this buffer contains components that may interfere with the assay chemistry, it is informative to blank the absorbances with a "water reference" (i.e., a zero-protein, water sample). Differences between the water reference and zero standard sample are then indicative of buffer effects.

Standard curve slope: The standard curve slope is directly related to assay accuracy and sensitivity. All else being equal, the steepest part of the curve is the most reliable. For most protein assays, the standard curve is steepest (i.e., has the greatest positive slope) in the bottom half of the assay range. In fact, the upper limit of an assay range is determined by the point at which the slope approaches zero; the line there is so flat that even a tiny difference in measured absorbance translates to a large difference in calculated concentration.

Measurement wavelength: The measurement wavelengths that are recommended for each protein assay method are optimal because they yield standard curves with maximal slope. This usually, but not always, corresponds to the absorbance maximum. (In certain circumstances, other considerations are also important in choosing the best possible measurement wavelength, such as avoiding interference from sample components that absorb at similar wavelengths). In fact, for most protein assays, depending on the precision required, acceptable results can be obtained using any measurement wavelengths within a certain range.

Yes, we offer a colorimetric and a fluorescent protease assay, Cat. Nos. 23263 and 23266, respectively.

The colorimetric kit uses fully succinylated casein as a substrate for the assay. Hydrolysis of this substrate in the presence of protease results in the release of peptide fragments with free amino-terminal groups. These peptides are reacted with trinitrobenzene sulfonic acid (TNBSA), followed by measurement of the absorbance increase that results from the formation of yellow colored TNB-peptide adducts.

The fluorescent includes fluorescein-labeled casein as a substrate for assessing protease activity in a sample by either fluorescence resonance energy transfer (FRET) with a standard fluorometer or fluorescence polarization (FP) with capable instrumentation. FTC-Casein is native casein that has been labeled using a large molar excess of fluorescein isothiocyanate (FITC). Fluorescence properties of this heavily-labeled, intact protein substrate change dramatically upon digestion by proteases, resulting in a measurable indication of proteolysis.

The colorimetric assay utilizes TNBSA which is measured at 450 nm. The fluorescent assay requires an instrument with fluorescein excitation and emission filters, 485/538 nm. 

• The Quant-iT™ and Qubit™ protein assaysare easy and accurate assays for the quantitation of protein samples ranging from 12.5 µg⁄mL to 5 mg⁄mL. These assays are highly selective for protein and exhibit very little protein-to-protein variation. Common contaminants, such as salts, solvents, or DNA (but not detergents) are well tolerated in these assays. The Qubit™ Protein Assay Kit is designed specifically for use with the Qubit™ Fluorometers, while the Quant-iT™ Protein Assay is designed to be used both on the Qubit™ fluorometer or other fluorometers and fluorescence plate readers. 
• The NanoOrange™ Protein Quantitation Kit contains a very sensitive and easy assay for protein quantitation, with detection as low as 10 ng/mL of protein in solution with a useful assay range of 10 ng/mL to 10 µg/mL. Common contaminants, such as salts, solvents, or DNA (but not detergents) are well tolerated in these assays. This fluorescent dye is suitable for use with spectrofluorometers and microplate readers. 
• The CBQCA Protein Quantitation Kit is a very sensitive assay for protein quantitation, with detection as low as 10 ng/mL of protein in solution with a useful assay range of 10 ng/mL to 150 µg/mL. CBQCA covalently modifies glutamine, asparagine and primary amines and requires cyanide for the reaction. This assay is tolerant of detergents, but amines, ammonium ions and reducing agents should be avoided. CBQCA is better suited for accurate quantitation of proteins in the presence of lipids, membrane fractions or detergents, and for lipoproteins and small peptides.
• The EZQ™ Protein Quantitation Kit provides accurate quantitation of protein samples in the range of 20 µg/mL to 5 mg/mL. The assay can be performed in the presence of detergents, urea, reducing agents, salts, solvents, dyes, and most other contaminants and is generally intended for samples prepared for 1D and 2D gel electrophoresis. 1 µL of each sample is spotted onto filter paper placed inside a specially-designed 96-well microplate, contaminants are washed away, and then the remaining bound proteins are stained with our proprietary fluorescent dye. The paper is then analyzed on a microplate reader or a laser scanner.

All the above assay kits come with either concentrated assay reagent and dilution buffer or a pre-diluted quantitation reagent and protein standards. The EZQ™ Protein Quantitation Kit also comes with a specially-designed 96-well microplate and filter paper that fits inside this microplate.

Fluorescent protein assays can be 10X more sensitive (EZQ™ Protein Quantitation Kit) to 100X more sensitive (NanoOrange™ Protein Quantitation Kit and CBQCA Protein Quantitation Kit) than colorimetric assays. They also have a more simplified workflow and can be performed in about an hour. 

Yes, the Quant-iT™ Protein Assay Kit manual has directions for this application. 

The EZQ™ Protein Quantitation Kit is the most tolerant of non-protein components. It is compatible with samples in SDS PAGE sample buffer, urea buffer and guanidine. After the protein component is bound to the paper filter, any tracking dyes, detergents and salts are removed by a methanol wash, so they are no longer present to affect protein quantitation.

Other protein assays that are compatible with detergents are colorimetric BCA assays, Detergent Compatible Bradford Assay Kit and 600 nm Protein Assay Kit.

The NanoOrange™ Protein Quantitation Kit and EZQ™ Protein Quantitation Kit are compatible with reducing agents.

We do not recommend that you use the Quant-iT™ Protein Reagent or Qubit™ Protein Reagent to quantify proteins in the presence of any detergents, surfactants, lipids or other chemicals that can either displace the dye from a hydrophobic region, disrupt lipid structure, or add a lipophilic/hydrophobic entity to the solution. The dye is environmentally sensitive; when it binds to hydrophobic pockets/domains, inserts into liposome lipid layers, or is dissolved in organic solvents, the fluorescence output increases relative to the fluorescence in aqueous solutions, potentially providing a higher background (assuming the liposome is not composed of anything that can quench fluorescence).

You may use the dye to quantitate purified protein and possibly a pure liposome sample (assuming that the solution the dye is dispersed in does not disrupt liposome structure), but it would be exceedingly difficult to quantify either as a mixed population.

The lowest protein size limit for these reagents has not been determined, although proteins as small as 6000 Da have been accurately quantitated. Quantitation accuracy of small peptides would likely be variable and dependent on the composition of the peptide. We would recommend using the CBQCA Protein Quantitation Kit for quantitation of small peptides. For quantitating peptide digest mixtures for mass spectrometry applications, we recommend using the Quantitative Colorimetric Peptide Assay (Cat. No. 23275) or Quantitative Fluorometric Peptide Assay (Cat. No. 23290).

Even though most of the non-protein components of the buffer are washed away as part of the staining procedure, the buffer composition affects the spread of the spot on the filter paper, which does affect the resulting signal and quantitation. Thus, for accurate quantitation, you need to prepare a standard curve for each general buffer your samples are in. Although the standard curve and sample buffers should be similar, there is some tolerance for buffers to be at different concentrations. If you have samples in different buffers, make an ovalbumin stock solution at 10 mg/mL in deionized water and then prepare serial dilutions with each buffer your samples are in, so that samples in different buffers have a buffer-specific standard curve.

For most fluorescent dyes, the EZQ™ Protein Quantitation Kit is the best choice and quantitation will not be affected by the presence of the dye. The one caution is if the protein is heavily labeled with a dye with an absorbance that overlaps the EZQ™ Protein Quantitation Reagent emission (approximately 600 nm), then the dye will quench the EZQ™ Protein Quantitation Reagent signal and not be compatible. A heavily labeled protein would have a pellet that is a very visible blue color; other color pellets or pellets with a pale blue color can be quantified with the EZQ™ Protein Quantitation kit. You can even label your proteins with a fluorescent dye that has similar excitation/emission properties as the EZQ™ Protein Quantitation Reagent; the EZQ™ Protein Quantitation Reagent produces a very strong signal that will dominate the combined signal of the two dyes.

You can image or scan the filter paper after the methanol wash (either wet or dried) before adding the EZQ™ Protein Quantitation Reagent using appropriate settings for your fluorescent dye, then scan again after performing the EZQ™ protein staining, at settings appropriate for the EZQ™ Protein Quantitation Reagent, to obtain a relative dye signal/protein amount quantitation.