Protein Quantification Discussion Examples | How to Interpret Calibration Curves, Absorbance, and Sources of Error

What Is Protein Quantification?

Protein quantification is an experiment used to numerically determine how much protein is contained in a sample.
In many quantification methods, proteins react with reagents to produce color, and the absorbance is measured.
Within the range where absorbance changes according to protein concentration, protein concentration can be determined from absorbance.

However, absorbance does not directly indicate the “amount of protein itself.”
A calibration curve must be prepared using a standard protein, and the relationship obtained must be used to convert the absorbance of an unknown sample into concentration.
Therefore, the accuracy of the calibration curve greatly affects the reliability of protein quantification.

Main Items to Include in the Results

In the results of protein quantification, organize the concentrations and absorbance values of the standard protein, the calibration curve, the absorbance of the unknown sample, the dilution factor, and the calculated protein concentration.
If multiple measurements were performed, showing the mean and variation also makes the discussion easier.

Main Items to Include in the Results

• Quantification method used
• Type of standard protein
• Standard protein concentration
• Absorbance of each standard solution
• Blank absorbance
• Absorbance after blank correction
• Calibration-curve equation
• Coefficient of determination R²
• Absorbance of the unknown sample
• Dilution factor
• Protein concentration of the unknown sample
• Mean of repeated measurements
• Standard deviation and variation
• Sources of error and points for improvement

Reference Experimental Values for Protein Quantification and Examples of Standard-Curve Analysis

Here, standard curves, absorbance values, unknown-sample concentrations, dilution factors, and measurement errors obtained in protein quantification experiments are organized as reference experimental values that are easy to discuss in reports.

In protein quantification, standard proteins of known concentration are measured to prepare a standard curve, and the absorbance of an unknown sample is applied to that standard curve to determine its concentration.
When considering the reliability of measured values, it is important to confirm the linearity of the standard curve, blank correction, dilution factor, measurement range, and variation among repeated measurements.

Reference Experimental Conditions

Example Measurement of Standard Proteins

The following is a reference example in which the relationship between protein concentration and absorbance was measured using BSA standard solutions.
Here, the measurement wavelength is 595 nm.

Absorbance increases as the concentration of the standard protein increases.
Because the standard deviations of the repeated measurements are small, the reproducibility of the standard-solution measurements is considered relatively good in this example.

Concept of Blank Correction

Even a blank containing no protein may show absorbance because of the color of the reagent itself or the effects of the cuvette or plate.
Therefore, the absorbance of the blank is subtracted from the absorbance of the standard solutions and unknown samples.

Blank-corrected absorbance = Measured absorbance − Blank absorbance

By performing blank correction, background absorbance originating from reagents and the measuring instrument can be subtracted, making it easier to handle absorbance changes derived from proteins.

Example Standard-Curve Equation

Suppose the following approximation equation is obtained when a standard curve is prepared using blank-corrected absorbance values.

Corrected absorbance = 0.00076 × Protein concentration (μg/mL) + 0.002

Solving this equation for concentration gives the following equation for determining the protein concentration of an unknown sample.

Protein concentration (μg/mL) = (Corrected absorbance − 0.002) ÷ 0.00076

If the unknown sample lies within the range of the standard curve, its concentration can be calculated using this equation.

Example Calculation of an Unknown Sample Concentration

Consider a case in which Unknown Sample A was diluted 10-fold and measured, giving a measured absorbance of 0.432 and a blank absorbance of 0.050.

Corrected absorbance = 0.432 − 0.050 = 0.382

Concentration after dilution = (0.382 − 0.002) ÷ 0.00076 = 500 μg/mL

Because this is the concentration after 10-fold dilution, the original sample concentration is as follows.

Original sample concentration = 500 × 10 = 5000 μg/mL = 5.0 mg/mL

Therefore, the protein concentration of Unknown Sample A is determined to be 5.0 mg/mL.

Example Measurement Results for Unknown Samples

Unknown Sample C lies on the high-concentration side of the standard curve, while Unknown Sample D lies on the low-concentration side.
Both values can be calculated, but adjusting the dilution factor so that the absorbance falls near the center of the standard curve allows a more reliable measurement.

Checking the Linearity of the Standard Curve

Before using a standard curve, it is necessary to confirm that it is linear within the measurement range.
On the high-concentration side, absorbance may become saturated and deviate from the straight line.

Even if the coefficient of determination is high, it does not necessarily mean that the entire range is accurate.
It is necessary to check whether the standard-solution points lie on the straight line and whether there is bias on the low- or high-concentration side.

Handling Values Outside the Calibration-Curve Range

If the absorbance of an unknown sample lies outside the standard-curve range, determining its concentration by extrapolation may cause a large error.

Outside the standard-curve range, there is less assurance that the relationship between absorbance and concentration remains valid.
Samples outside the range should be diluted or concentrated so that they fall within the measurement range and then remeasured.

Differences in Final Concentration Depending on Dilution Factor

Even when the concentration determined from the same absorbance is the same, the original sample concentration changes depending on the dilution factor used during measurement.

In a report, clearly state whether the concentration determined from the standard curve is the “concentration of the diluted sample measured” or the “concentration converted back to the original sample.”

Example of an Outlier

If only one measurement deviates greatly during repeated measurements, possible causes include bubbles, insufficient mixing, pipetting errors, or contamination of the cuvette.

If only the third measurement of Unknown Sample B is high, bubbles or contamination may have caused the absorbance to appear high.
If an outlier is excluded, the reason should be clearly stated and remeasurement should be performed if possible.

Effect of Errors in Standard-Solution Preparation

If the concentrations of standard solutions are prepared incorrectly, the entire standard curve shifts and the unknown-sample concentrations are also calculated incorrectly.

Error Caused by the Sample’s Own Color or Turbidity

If an unknown sample has color or turbidity, absorbance other than that produced by the protein reaction is included in the measured value.
In this case, correction can be facilitated by using a sample blank.

In colored or turbid samples, failure to subtract the sample blank may cause the protein concentration to be overestimated.

Example Comparison of Concentration Differences Among Measurement Methods

Even for the same sample, the protein concentration obtained may differ slightly depending on the measurement method.

Protein quantification methods are based on different reaction principles.
Therefore, even if the values are not exactly the same, it is important to discuss the results while considering the characteristics of each measurement method.

Examples of the Effects of Interfering Substances

When interfering substances are present, measured values may not reflect protein concentration alone.
Possible measures include preparing standard solutions in the same buffer as the sample, using a sample blank, or confirming the result with another method.

Example of Calculating Relative Standard Deviation From Repeated Measurements

To evaluate measurement variation, the mean, standard deviation, and relative standard deviation may be calculated.

Mean = 5.01 mg/mL

Standard deviation = 0.05 mg/mL

Relative standard deviation (RSD) = 0.05 ÷ 5.01 × 100 = 1.0%

When the RSD is small, the reproducibility of repeated measurements is considered relatively good.

Example of How to Write the Results

A standard curve for protein quantification was prepared using BSA standard solutions.
The blank absorbance was 0.050, and this value was subtracted from the mean absorbance of each standard solution for correction.
As a result, absorbance increased with increasing protein concentration over the range of 0–1000 μg/mL, and the standard curve was expressed as A = 0.00076C + 0.002.

When Unknown Sample A was diluted 10-fold and measured, the measured absorbance was 0.432.
The absorbance after blank correction was 0.382, and substitution into the standard curve gave a concentration after dilution of 500 μg/mL.
Because the original sample had been diluted 10-fold, the original protein concentration was 5.0 mg/mL.

In Unknown Sample C, the corrected absorbance was 0.665 and was located on the high-concentration side of the standard curve.
In this case, although the value lies within the measurement range, it may be affected by saturation of color development or deviation from linearity.
For more accurate measurement, it is desirable to further dilute the sample and remeasure at an absorbance near the center of the standard curve.

Points for Connecting the Results to the Discussion

In a discussion of protein quantification, it is important to explain not only the calculated concentration but also the reliability of the standard curve, the measurement range, and the sources of error.

• Has a standard curve been prepared from the standard solutions?
• Has blank correction been performed to subtract background absorbance?
• Can the concentration be calculated by substituting the absorbance of the unknown sample into the standard-curve equation?
• Has the original sample concentration been restored by multiplying by the dilution factor?
• Has it been confirmed that the absorbance of the unknown sample lies within the standard-curve range?
• Can the possibility of absorbance saturation and reduced linearity on the high-concentration side be discussed?
• If an outlier is present, can bubbles, insufficient mixing, pipetting errors, contamination of the cuvette, and similar causes be explained?
• Can it be discussed that errors in preparing the standard solutions affect the entire standard curve?
• Can it be explained that the sample’s own color, turbidity, and interfering substances affect absorbance?
• Can differences in results among measurement methods be explained in relation to differences in reaction principles?

Example Discussion

In this experiment, a standard curve was prepared using BSA standard solutions, and the protein concentration in an unknown sample was determined.
Absorbance increased as the concentration of the standard solutions increased, and a good linear relationship was obtained over the range of 0–1000 μg/mL.
Therefore, when the absorbance of an unknown sample falls within this range, the concentration can be determined using the standard curve.

For Unknown Sample A, the measured absorbance of the 10-fold diluted solution was 0.432.
Subtracting the blank absorbance of 0.050 gave a corrected absorbance of 0.382, and the concentration after dilution was determined to be 500 μg/mL from the standard curve.
Multiplying this value by the dilution factor of 10 gave an original sample protein concentration of 5.0 mg/mL.
Therefore, both blank correction and dilution-factor correction are important in concentration calculations.

Possible sources of measurement error include errors in preparation of the standard solutions, variation in pipetting, differences in reaction time, insufficient mixing, bubbles, and contamination of the cuvette.
In absorbance measurements in particular, bubbles and turbidity may scatter light and produce absorbance values higher than the actual values.
In addition, if the concentrations of the standard solutions are prepared incorrectly, the slope of the standard curve changes and affects all calculations of unknown-sample concentrations.

When absorbance lies on the high-concentration side, as in Unknown Sample C, the color reaction may become saturated and the linear relationship between absorbance and concentration may break down.
If values outside the standard-curve range are extrapolated, the concentration may be greatly misestimated.
Therefore, unknown samples should be diluted as necessary and adjusted so that they are measured near the center of the standard curve.

Furthermore, if the sample itself has color or turbidity, absorbance unrelated to the protein reaction may be included in the measured value.
In this case, a sample blank must be measured and the absorbance originating from the sample itself subtracted.
In addition, because interfering substances such as surfactants, reducing agents, high concentrations of salts, and nucleic acids may affect measured values, it is important to select an appropriate quantification method according to the sample composition.

Summary

In protein quantification, a standard curve is prepared using a standard protein, and the concentration of an unknown sample is determined from its absorbance.
To obtain an accurate concentration, blank correction, dilution-factor correction, measurement within the standard-curve range, and confirmation of repeated measurements are important.

This reference example covered standard curves, blank correction, calculation of unknown-sample concentrations, linearity, handling of values outside the calibration-curve range, dilution factors, outliers, errors in standard-solution preparation, sample blanks, interfering substances, and differences among measurement methods.
In a report, it is useful to clearly show the concentration-calculation procedure and discuss both the reliability of the measured values and the sources of error.

What Is a Calibration Curve?

A calibration curve is a graph showing the relationship between a standard sample of known concentration and the measured value.
In protein quantification, the standard protein concentration is often plotted on the horizontal axis and absorbance on the vertical axis.
Within the range where absorbance is proportional to concentration, the measurement points approach a straight line.

By applying the absorbance of an unknown sample to the calibration curve, the protein concentration of the unknown sample can be determined.
However, if the absorbance of the unknown sample lies outside the calibration-curve range, the result becomes an extrapolation and reliability decreases.
In that case, the sample must be diluted or remeasured.

Discussion of Calibration-Curve Linearity

If the calibration curve has good linearity, the relationship between standard protein concentration and absorbance is considered stable.
When the coefficient of determination R² of the approximation line is close to 1, the measurement points are considered to fit the straight line well.
However, a high R² alone does not necessarily mean that all measurements are correct.

On the low-concentration side, absorbance is small and the effects of blank correction and measurement noise may become large.
On the high-concentration side, the color reaction or absorbance measurement may become saturated and deviate from the straight line.
Therefore, it is important to confirm which concentration range can be used as a linear range in the calibration curve.

Relationship Between Absorbance and Protein Concentration

In protein quantification, as the protein concentration increases, the color reaction often becomes stronger and absorbance becomes larger.
This is because the intensity of the color produced by the reaction between proteins and the reagent increases according to the amount of protein.

However, absorbance is proportional to concentration only within a certain range.
If the concentration is too high, the reagent may become insufficient, the color reaction may become saturated, or the absorbance may exceed the linear range of the measuring instrument.
In such cases, directly converting absorbance to concentration causes a large error.

Discussion of Blank Correction

Blank correction is the operation of subtracting absorbance caused by reagents and solvents other than proteins.
A blank is often prepared by adding the same reagents under conditions without protein.
Blank correction reduces the effects of absorbance originating from the reagents, cuvette, and solvent.

If blank correction is insufficient, the absorbance of the unknown sample may be overestimated or underestimated.
Particularly for low-concentration samples, even a slight deviation in the blank can have a large effect on concentration calculations.

Concept of Determining the Concentration of an Unknown Sample

The protein concentration of an unknown sample is determined by substituting its absorbance into the calibration-curve equation.
For example, if the calibration-curve equation is y = ax + b, the absorbance of the unknown sample is substituted for y and the concentration is determined as x.
If the sample was diluted before measurement, the original sample concentration is then determined by multiplying by the dilution factor.

Original sample concentration = Measured sample concentration × Dilution factor

In a report, it is important to distinguish whether the concentration determined from the calibration curve is the “concentration of the diluted measurement sample” or the “concentration of the original sample.”

Discussion of the Dilution Factor

In protein quantification, when the concentration of an unknown sample is too high, the sample is diluted so that it falls within the calibration-curve range.
Dilution allows the absorbance to be brought into an appropriate range, but the dilution factor must be correctly reflected in the calculation.

If the dilution factor is incorrect, the final protein concentration will deviate greatly.
For example, if the concentration obtained from a 10-fold diluted sample is written directly as the original sample concentration, the value will be one-tenth of the actual concentration.

When the Unknown Sample Is Outside the Calibration-Curve Range

If the absorbance of an unknown sample is higher than that of the highest concentration on the calibration curve, determining the concentration directly results in extrapolation.
Extrapolation assumes that the linear relationship of the calibration curve continues outside the measured range, so reliability decreases.
On the high-concentration side, the color reaction may also be saturated.

Conversely, if the absorbance of the unknown sample is too low, the effects of the blank and measurement noise become large.
In this case, possible measures include increasing the sample amount, concentrating the sample, or using a more sensitive method.

Differences Between Standard Proteins and Unknown Samples

In protein quantification, proteins such as BSA may be used as standard proteins.
However, depending on the quantification method, the intensity of color development may differ depending on the type of protein.
In other words, even at the same mass concentration, the absorbance of a standard protein and an unknown sample may not be exactly the same.

In the Bradford assay, the dye binds mainly depending on specific amino acid residues and the properties of proteins, so the response may differ among protein types.
In the UV absorption method, differences in the content of aromatic amino acids such as tryptophan and tyrosine affect absorbance.

Discussion of the Bradford Assay

The Bradford assay is a quantification method that uses the change in absorbance caused when Coomassie Brilliant Blue dye binds to proteins.
Because it can be measured in a relatively short time and has high sensitivity, it is commonly used in biochemical experiments.

In the Bradford assay, the ease with which the dye binds may differ depending on the type of protein.
In addition, some substances such as surfactants may affect the measurement.
Because the linear range of the calibration curve is limited, dilution of unknown samples is important.

Discussion of the Lowry Method and BCA Assay

The Lowry method and BCA assay are quantification methods that develop color by using reactions such as reduction of copper ions by proteins.
These methods are highly sensitive and widely used, but may be affected by reducing agents, chelating agents, surfactants, buffer components, and similar substances.

If DTT, β-mercaptoethanol, EDTA, strong surfactants, or similar substances are present in the sample, they may affect the color reaction and cause the protein concentration to be overestimated or underestimated.
In a report, it is useful to include the components of the sample buffer in the discussion.

Discussion of the UV Absorption Method

In the UV absorption method, protein concentration is determined using the fact that proteins absorb near 280 nm.
This absorption mainly originates from aromatic amino acids such as tryptophan and tyrosine.
Therefore, absorbance at 280 nm differs greatly depending on the type of protein.

In addition, nucleic acids such as DNA and RNA absorb near 260 nm, so contamination with nucleic acids may cause the protein concentration to be overestimated.
In the UV absorption method, it is important to consider the purity of the sample and the effects of contaminants.

Effects of Interfering Substances in the Sample

In protein quantification, components in the sample may affect the color reaction or absorbance measurement.
Representative interfering substances include surfactants, reducing agents, chelating agents, salts, high-concentration buffers, organic solvents, and nucleic acids.
When these substances are present, absorbance may change independently of the amount of protein.

Errors Caused by Pipetting

In protein quantification, small volumes of standard solutions, unknown samples, and color reagents must be dispensed accurately.
Errors in micropipette operation cause deviations in concentration and reagent volume and affect absorbance.
Particularly when preparing a calibration curve, errors in the concentration series of standard solutions affect the entire approximation line.

Possible causes include liquid remaining at the tip, aspirating air bubbles, using the wrong plunger position, and difficulty accurately aspirating viscous samples.
In small-volume operations, even a small volume error can have a relatively large effect.

Errors Caused by Reaction Time

In color reactions used for protein quantification, reaction time may affect absorbance.
If the reaction time is too short, color development does not proceed sufficiently and absorbance becomes small.
Conversely, if the reaction time is too long, color development may proceed too far or stability may decrease, making comparisons between standard solutions and unknown samples inaccurate.

If the reaction times differ between standard solutions and unknown samples, they cannot be compared under the same conditions.
Therefore, it is important to standardize the time from the start of the reaction to measurement.

Errors Caused by Contamination of Cuvettes and Plates

In absorbance measurements, contamination, scratches, fingerprints, and bubbles on cuvettes or microplates affect the measured values.
If contamination or bubbles are present in the optical path, light transmission is hindered and absorbance may appear higher than the actual value.
Turbidity and precipitates in the sample also cause light scattering and affect absorbance.

Discussion When Absorbance Is Too High

If the absorbance of an unknown sample is too high, the protein concentration may exceed the calibration-curve range.
At high absorbance, the linearity of the measuring instrument decreases and the proportional relationship with concentration is more likely to break down.
Light scattering caused by sample turbidity or precipitation and color development caused by interfering substances may also increase absorbance.

Discussion When Absorbance Is Too Low

If the absorbance of an unknown sample is too low, possible causes include a low protein concentration, excessive dilution of the sample, insufficient progression of the color reaction, and protein degradation or precipitation.
At low absorbance, the relative effects of blank correction and measurement noise become larger.

Causes of Overestimating Protein Concentration

Causes of overestimating protein concentration include insufficient blank correction, contamination of the cuvette, sample turbidity, color development caused by interfering substances, errors in preparing standard-solution concentrations, and errors in the dilution factor.
In particular, if absorbance becomes high because of factors other than proteins, the concentration determined from the calibration curve also becomes high.

Causes of Underestimating Protein Concentration

Causes of underestimating protein concentration include protein degradation, precipitation, insufficient extraction, inhibition of the color reaction, excessive dilution of the sample, insufficient reaction time, and deterioration of the standard solution or reagent.
If proteins have precipitated, measuring only the supernatant gives a value lower than the actual total amount of protein.

Discussion of Variation in Repeated Measurements

In protein quantification, the same sample may be measured multiple times and the mean value determined.
If repeated measurements agree well, the reproducibility of the operation and measurement is considered high.
On the other hand, if variation is large, possible causes include pipetting, insufficient mixing, differences in reaction time, bubbles, and differences in plate position.

Discussion When Calibration-Curve Points Deviate

Some standard points on a calibration curve may deviate greatly from the approximation line.
Possible causes include dilution errors in the standard solutions, pipetting errors, insufficient mixing of the color reagent, bubbles, and errors in reading absorbance.
If an outlier is excluded, the reason must be based on the experimental operation rather than simply because the value is inconvenient.

Discussion of the Calibration-Curve Intercept

The intercept of the calibration curve corresponds to the absorbance when the protein concentration is zero.
If blank correction is appropriate, the intercept is expected to be close to zero.
However, if there is reagent-derived absorbance, contamination of the cuvette, or insufficient blank correction, the intercept may deviate from zero.

Effects of Protein Denaturation and Degradation

Proteins may denature or degrade because of temperature, pH, enzymatic degradation, freeze-thaw cycles, organic solvents, and other factors.
When denaturation or degradation occurs, reactivity with the color reagent may change or proteins may precipitate and become difficult to measure.
As a result, the quantified value may differ from the actual amount of protein.

Buffer Conditions of the Standard Curve and Unknown Sample

If the buffer conditions of the standard protein and unknown sample differ, differences may occur in the color reaction or absorbance.
For example, if only the unknown sample contains high concentrations of salts, surfactants, reducing agents, or chelating agents, it may not be possible to compare it under the same conditions as the standard curve.

For more accurate quantification, the standard protein may be dissolved in the same buffer as the unknown sample when preparing the calibration curve.
This makes it easier to standardize the effects of buffer components between the standard solutions and unknown sample.

When the Results Can Be Considered Good

Protein quantification results can be considered good when the calibration curve shows good linearity, the absorbance of the unknown sample falls within the calibration-curve range, and variation among repeated measurements is small.
It is also important that blank correction be appropriate and that the sample dilution factor and buffer conditions be correctly taken into account.

Example Discussion When the Experiment Did Not Go Well

If protein quantification does not go well, the causes are considered from results such as a non-linear calibration curve, an unknown sample outside the range, variation in repeated measurements, excessively high or low absorbance, or a large blank.
Organizing the causes into the calibration curve, sample, reagents, operation, and measuring instrument makes the discussion easier.

How to Write Points for Improvement

In a discussion of protein quantification, including not only sources of error but also points for improvement makes the report easier to organize.
Improvements are easier to organize when divided into standard-solution preparation, pipetting, reaction conditions, measurement conditions, and sample pretreatment.

Improvements to Standard Solutions and the Calibration Curve

• Prepare standard protein concentrations accurately
• Prepare the dilution series carefully
• Mix each standard solution thoroughly
• Confirm the linear range of the calibration curve
• If an outlier appears, check the operation record

Improvements to Sample Measurement

• Dilute unknown samples so that they fall within the calibration-curve range
• Correctly reflect the dilution factor in the calculation
• Check the sample for turbidity or precipitates
• Perform centrifugation or filtration when necessary
• Match the buffer conditions of the standard solutions and unknown samples

Improvements to Operations and Measurements

• Use the micropipette accurately
• Avoid bubbles in the tip
• Keep the color-development reaction time consistent
• Perform blank correction appropriately
• Avoid contamination of cuvettes and plates
• Perform multiple measurements and use the mean value

Difference Between a Superficial Discussion and a Good Discussion

In a discussion of protein quantification, simply writing that “the concentration was determined from the calibration curve” or “there was an error” results in a superficial discussion.
Relating calibration-curve linearity, the absorbance range of the unknown sample, blank correction, dilution factor, interfering substances, and operational errors produces a more persuasive discussion.

Examples of Expressions That Can Be Used in Reports

The following expressions can be used when writing the results and discussion of protein quantification.
Adjust the necessary parts according to your own experimental results.

• A calibration curve was prepared from the standard protein concentrations and absorbance values.
• Because the calibration curve showed good linearity, protein concentration could be determined from absorbance within the measurement range.
• Because the absorbance of the unknown sample fell within the calibration-curve range, the concentration could be calculated without extrapolation.
• Because the unknown sample was diluted before measurement, the dilution factor was taken into account when determining the original sample concentration.
• Blank correction was used to subtract absorbance originating from the reagents and solvent.
• On the high-concentration side, the color reaction may become saturated and the proportional relationship between absorbance and concentration may break down.
• Surfactants or reducing agents in the sample may have affected the color reaction and caused errors in the quantified value.
• Contamination or bubbles in the cuvette can cause absorbance to appear high.
• If reaction time differs among samples, the extent of color development changes and variation in absorbance occurs.
• If the properties of the standard protein and unknown sample differ, color intensity may differ even at the same concentration.

Points to Check When Discussing Protein Quantification

Checking the following points before writing the report makes the discussion easier to write.

• Have you stated the quantification method used?
• Have you written the type of standard protein?
• Have you shown the calibration-curve equation and R²?
• Have you confirmed the linear range of the calibration curve?
• Have you confirmed that the absorbance of the unknown sample lies within the calibration-curve range?
• Have you performed blank correction?
• Have you correctly reflected the dilution factor in the calculation?
• Have you discussed variation among repeated measurements?
• Have you considered errors caused by pipetting and reaction time?
• Have you considered the effects of interfering substances?
• Have you considered differences between the standard protein and the unknown sample?
• Do the points for improvement correspond to the sources of error?

Summary

In protein quantification, a calibration curve is prepared using a standard protein, and the protein concentration is determined from the absorbance of an unknown sample.
Good linearity of the calibration curve and an unknown-sample absorbance that falls within the calibration-curve range are important for reliable quantification.
If the unknown sample is diluted before measurement, the dilution factor must always be taken into account when determining the original sample concentration.

Major sources of error in protein quantification include errors in preparing standard solutions, pipetting, insufficient blank correction, differences in color-development reaction time, contamination of cuvettes or plates, interfering substances in the sample, and protein denaturation or precipitation.
In addition, if the properties of the standard protein and unknown sample differ, the intensity of color development may differ even at the same concentration.

In a report, rather than simply writing that “the concentration was determined from the calibration curve,” discuss calibration-curve linearity, measurement range, blank correction, dilution factor, interfering substances, and operational errors in relation to one another.
Explaining which factors may cause the concentration to be estimated too high or too low produces a more persuasive discussion of protein quantification.