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. This comparative method for determining the concentration of an "unknown" is conceptually simple and straightforward. However, its implementation in an assay protocol is complicated by pipetting and dilution steps, evaluation of replicates, blank-corrections and other factors. These steps frequently cause confusion with regard to the calculations that are necessary to obtain a final determination.

Preparation of standards for developing a standard curve

The following tables provide information to prepare a set of protein standards for a standard curve for common BCA assay and Bradford assays. To proceed dilute the contents of a 1 mL BSA standard (2 mg/mL) into several clean vials, preferably using the same diluent as the test samples.

BCA Protein Assay standard curve

Table 1. Microplate or test tube.

Vial Volume of diluent Volume and source of BSA Final BSA concentration
A 0 300 µL of stock 2,000 µg/mL
B 125 µL 375 µL of stock 1,500 µg/mL
C 325 µL 325 µL of stock 1,000 µg/mL
D 175 µL 175 µL of vial B dilution 750 µg/mL
E 325 µL 325 µL of vial C dilution 500 µg/mL
F 325 µL 325 µL of vial E dilution 250 µg/mL
G 325 µL 325 µL of vial F dilution 125 µg/mL
H 400 µL 100 µL of vial G dilution 25 µg/mL
I 400 µL 0 0 µL/mL = blank

Table 2. Enhanced test tube for BCA Protein Assay.

Vial Volume of diluent Volume and source of BSA Final BSA concentration
A 700 µL 100 µL of stock 250 µg/mL
B 400 µL 400 µL of vial A dilution 125 µg/mL
C 450 µL 300 µL of vial B dilution 50 µg/mL
D 400 µL 400 µL of vial C dilution 25 µg/mL
E 400 µL 100 µL of vial D dilution 5 µg/mL
F 400 µL 0 0 µL/mL = blank

Bradford Protein Assay standard curve

Table 3. Microplate or test tube.

Vial Volume of diluent Volume and source of BSA Final BSA concentration
A 0 300 µL of stock 2,000 µg/mL
B 125 µL 375 µL of stock 1,500 µg/mL
C 325 µL 325 µL of stock 1,000 µg/mL
D 175 µL 175 µL of vial B dilution 750 µg/mL
E 325 µL 325 µL of vial C dilution 500 µg/mL
F 325 µL 325 µL of vial E dilution 250 µg/mL
G 325 µL 325 µL of vial F dilution 125 µg/mL
H 400 µL 100 µL of vial G dilution 25 µg/mL
I 400 µL 0 0 µL/mL = blank

Table 4. Microplate or test tube for dilute samples.

Vial Volume of diluent Volume and source of BSA Final BSA concentration
A 237 µL 3 µL of stock 25 µg/mL
B 495 µL 5 µL of stock 20 µg/mL
C 397 µL 3 µL of stock 15 µg/mL
D 250 µL 250 µL of vial B dilution 10 µg/mL
E 200 µL 200 µL of vial D dilution 5 µg/mL
F 150 µL 150 µL of vial E dilution 2.5 µL/mL
G 500 µL 0 0 µL/mL = blank

Modified Lowry Protein Assay standard curve

Table 5. Microplate or test tube.

Vial Volume of diluent Volume and source of BSA Final BSA concentration
A 250 µL 750 µL of stock 1,500 µg/mL
B 500 µL 500 µL of vial A dilution 1,000 µg/mL
C 0.5 mL 500 µL of vial A dilution 750 µg/mL
D 500 µL 500 µL of vial B dilution 500 µg/mL
E 500 µL 500 µL of vial D dilution 250 µg/mL
F 500 µL 500 µL of vial E dilution 125 µg/mL
G 800 µL 200 µL of vial F dilution 25 µg/mL
H 1,000 µL 0 0 µL/mL = blank

Principles of standard curve

1. 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 all four of the follow conditions are met:

  • Samples are dissolved in the same buffer
  • The same lot and stock of assay reagent is used for all samples
  • All samples are mixed and incubated at the same time and temperature
  • No pipetting errors are introduced

Of course, because of differences in the chemistry of protein assay methods, different proteins will generate different absorbance values even at the same concentration. This is called "protein-protein variation" or "protein uniformity" and is discussed more fully in other protein methods articles.

Pierce BCA Protein assay standard curve with BGG and BSA

Figure 1. Example standard curves for the Thermo Scientific Pierce BCA Protein Assay Kit. Eight concentrations of bovine serum albumin (BSA) and bovine gamma globulin (BGG) were assayed. The response values (absorbances) were plotted and a best-fit line drawn through the points. If unknown samples had been tested at the same time, their concentrations could be determined by reference to the one of these standard curves.

2. Units in equals 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). For example, if the standards are expressed as micrograms per milliliter (µg/mL), then the value for the unknown sample, which is determined by comparison to the standard curve, is also expressed as micrograms per milliliter.


Common misconceptions

1. Identically assayed samples are directly comparable

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. Consider a simple example in which the Coomassie Plus Protein Assay Kit (Part No. 23236) is used to assay two protein samples: a test sample whose concentration is not known, and a standard whose concentration is 1 mg/mL (= 1000 µg/mL). (Ordinarily, an entire set of standards is necessary to establish a response curve, but this is a simplified example.)

In the microplate protocol (see figure), one adds 10 µL of sample (test or standard) and 300 µL of assay reagent per well. Because 10 µL of the standard sample is added to a well, there is 0.010 mL x 1,000 µg/mL = 10 µg of protein in the well. If the assay results in the test sample having the same final absorbance as the standard sample, then the conclusion is that the test sample contains the same amount of protein as the standard sample. Because there was 10 µg of standard per well, one could report the determined concentration of test sample as 10 µg/well. However, 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. Because the original standard was 1000 µg/mL, the test sample that produced the same absorbance in the assay also must be 1000 µg/mL.

Diagram of protein assay steps.

Figure 2. Diagram of protein assay steps. If standard (top row) and unknown (bottom row) samples are dispensed and mixed with the same amount of assay reagent, then they are directly comparable. If the absorbances of the final solutions (green) are identical, then the concentration of the unknown sample is determined to be 1 mg/mL. The amount of protein in the assay well (middle) and the concentration in assay reagent (right) are irrelevant.

2. Misconception: I need to know the protein concentration in the assay reagent

Furthermore, it is neither necessary nor helpful to know the protein concentration as it exists when diluted in assay reagent. In the above example, because the 10 µg standard was diluted to 310 µL after adding 300 µL of assay reagent, the final concentration in the well is 10 µg/310 µL = 0.0323 µg/µL = 32.3 µg/mL. Therefore, one could report the determined concentration of test sample as 32.3 µg/mL. However, 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. Because the original standard was 1000 µg/mL, the test sample that produced the same assay absorbance also must be 1000 µg/mL.


Applying dilution factors

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. Continuing with the same example, suppose that the original protein sample is actually known to be approximately 5 mg/mL. This is too concentrated to be assayed by the Coomassie Plus Protein Assay Kit, whose assay range in the standard microplate protocol is 100-1500 µg/mL. However, one 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 one 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.


Curve-fitting and interpolation

Unlike the example described above, most assays use an entire set of protein standards whose concentrations span the effective assay range (e.g., 100-1500 µg/mL). Rarely, if ever, will the test sample produce an assay response that corresponds exactly to one of the specific standard samples. Therefore, a method is needed to calculate or interpolate between the standard sample points.

How to use a standard curve

Figure 3. How to use a standard curve. Absorbance values for different known concentrations of a protein standard are plotted (diamonds).

If a test sample produces an absorbance of 0.6, how does one use the standard curve to calculate the protein concentration?

Most plate readers and spectrophotometers have associated software that automatically plots a best-fit (linear or curvilinear) regression line through the standard points, interpolates the test samples on that regression line, and reports the calculated value. It is important to choose an appropriate curve-fitting algorithm because the mathematical formula describing the fitted curve will be used to calculate the concentration of the test sample.

The following figures illustrate how different curve-fitting algorithms affect the accuracy of protein assay calculations. Few, if any, protein assays are perfectly linear over the entire useful assay range. As long as the appropriate curve-fit is used, an assay does not need to be linear to be accurate. If curve-fitting must be done manually, a point-to-point fit will usually be more accurate than a linear fit to the entire range of standard points (see figure). A "point-to-point" fit is a linear fit between each successive pair of points.

Comparison of point-to-point and linear-fit standard curves

Figure 4. 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.

Protein concentration determination using a standard curve

Figure 5. Comparison of point-to-point and best-fit standard curves. Interpolation and calculation for an unknown sample having absorbance 0.6 results in slightly different protein concentration values. In this case, the best-fit curve provides a more accurate calculation than the point-to-point curve.


Other factors affecting assay accuracy and precision

Replicates

Several factors affect protein assay accuracy and precision. 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 the technician's pipetting precision. If replicates are used, curve-fitting is done on the average value (minus obvious outliers).

Blank correction

Published graphs of standard curves (as in the figures on this page) usually show the line going through the origin (0,0). Although visually appealing, this is irrelevant to the calculations. Most protein assay working reagents have absorptivity at the detection wavelength (i.e., they have positive absorbance even when there is no protein present, see figures below).

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.

Pierce BCA Protein assay standard curve with BGG and BSA

Figure 6. Standard curves. Typical standard curves for bovine serum albumin (BSA) and bovine gamma globulin (BGG) in the BCA Protein Assay. Note that the value of the zero protein blank has been subtracted from all standards (and would also be subtracted from any unknowns) in order to eliminate absorbance contributed by the sample buffer, thus the zero standard is located at the origin of the graph.

Standard curve slope and measurement wavelength

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.

However, the very lowest part of the assay range, while having the steepest slope, is not always optimal because random errors and interfering substances have greater relative effects in samples containing very small amounts of protein. Therefore, the clearest results are usually obtained with test samples that are pre-diluted so that they correspond to the lower-middle portion of the assay range. Many technicians test samples at two or three dilutions to ensure that at least one of them "lands" in this part of the assay range.

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. The following figures illustrate this point (see Tech Tip #25 for details).

Pierce BCA protein assay absorbance spectra

Figure 7. BCA protein assay absorbance spectra. Scans of eight BSA standards (0 to 2000 µg/mL) tested with the Thermo Scientific Pierce BCA Protein Assay Kit (Part No. 23225). The thick line is the 2000 µg/mL sample. The optimum measurement wavelength for this assay is 562 nm.

Pierce Coomassie Bradford protein assay absorbance spectra

Figure 8. Coomassie (Bradford) protein assay absorbance spectra. Scans of eight BSA standards (0 to 2000 µg/mL) tested with the Thermo Scientific Pierce Coomassie Plus Protein Assay Reagent (Part No. 23236). The thick line is the 2000 µg/mL sample. The optimum measurement wavelength for this assay is 595 nm. An inverse standard curve can be generated by measuring at 465 nm. Notice that the baseline absorbance of this reagent is ~0.4 at 595 nm (measured in a 1 cm cuvette).

Interfering substances

The possible effects of interfering substances were not discussed in this article because the assumption was that all protein samples were treated exactly the same, including the buffers in which the proteins were dissolved. In this situation, any interference caused by components of the buffers is exactly the same for both test and standard samples. Nevertheless, interference by non-protein substances in the samples that block or contribute to the assay color reaction is an important issue for any protein assay system. Refer to related articles and documents for further discussion this topic.


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