Bradford Assay
Title:
Bradford Assay: Creating a Standard Curve and Determining Unknown Protein Concentrations
Introduction:
In biochemistry the ability to determine the quantity of a certain substance in a solution is extremely important. Often protein concentration within a biological organism is essential in determining the function or condition of that organism. If the protein concentration in an environment is increased or decreased it can have detrimental side effects. If there is a decrease in hemoglobin (a protein found within blood), oxygen will not be able to be transported throughout the body. This can lead to cell death where the oxygen does not reach; therefore knowing that the body has the correct concentration of hemoglobin is important. …show more content…
The protein concentration can be measured using various methods such as Mass Spectrometry, Western blots, and colorimetric assays. Colorimetric assays determine the solute concentration of a solution based on the depth of color of the solution. For example a solution that has a deeper color will result in a higher concentration of solute. Since colorimetric assays rely on color concentration, a dye must be added to the solution so the absorbance can be taken. There have been many adaptations of colorimetric assays that test for various substances, such as the Bradford assay for total protein.
The Bradford assay for total protein is a colorimetric assay that measures the total concentration of protein in a solution. Since the Bradford assay is a colorimetric assay a dye must be used to color the solution for light to absorb in the substance. The dye used in a Bradford Assay is known as Coomassie Blue G-250, which will bind to protein when in an acidic solution1. The absorbance of samples with known concentrations can be taken and, using Beer-Lamberts Law, a standard curve can be produced. The standard curve will produce an equation in the form of, y = mx + b; which can be used to determine the concentration of an unknown sample. However, the Bradford assay is only effective between a concentration range of 1.2 μg/mL to 10 μg/mL. Therefore the samples must be made to be within this range.
The experiment aims to construct a standard curve using the Bradford Assay and varying concentrations of a known protein, bovine serum albumin (BSA). The absorbance will be taken at 595nm, due to the appropriate absorbance range for BSA. Using the standard curve produced by BSA, the concentrations of two unknown proteins will be determined.
Methods:
Preparation of Standard Curve:
A stock solution of bovine serum albumin (BSA) with a concentration of 1 mg/mL was diluted down to five solutions (in triplicate) with concentrations of 0 μg/mL, 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL, and 10 μg/mL. An equivalent volume (in ratio to the entire solution) of dye, Coomassie Blue G-250, was then added to the solution. The solutions containing the dye were then incubated for ten minutes. The absorbances of these solutions were then taken using a Perkin- Elmer Lamda Bio spectrometer, at an absorbance of 595 nm. A graph that plotted average absorbance verses concentration was created and a least-square linear regression line was obtained to determine the standard curve formula; in form of y = mx + b, when y is absorbance, m is slope, b is y-intercept, and x is concentration.
Determination of Unknown Protein:
Two unknown protein samples were selected at random, Unknown D (UD) and Unknown C (UC). The unknowns were then diluted down to various, random concentrations. The diluted solutions were combined with equivalent volumes of Coomassie Blue G-250 and left to incubate for ten minutes. The absorbances were then taken at 595 nm. The samples were diluted until the measured absorbance fell within range of the standard curve.
Results:
Five sample solutions of BSA with concentrations of 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL, 10 μg/mL were made, in triplicate, using 1 mg/mL BSA (table 1).
Calculations are as followed:
Sample (Tube) A was prepared with pure dH2O since the concentration of protein was 0 μg/mL. Upon addition of Coomassie Blue G-250 and the completion of the incubation period it was noticeable that solutions of higher concentration had a deeper color blue opposed to those with a lower concentration. The absorbance range for the standard curve (figure 1) was determined to be between 0.047A and 0.317A (table 1). This was determined to be the range in which the unknown absorbance had to fall.
Unknown C was diluted by a factor of 1 μL UC/1001 μL dH¬¬2O and had an absorbance of .0754A. The resulting solution had a light blue coloration. The dilution was decreased to 1 μL UC/501 μL dH2O and had an absorbance of 0.089A (table 2). This solution had a slightly darker blue coloration, however it was still a light blue in color. The absorbance was taken two more times at this dilution resulting in an average absorbance of 0.097A (table 2). The concentration of Unknown C was determined to be 2.655 μg/mL using the formula, which was attained from the standard curve. The correct concentration of unknown C was found to be 1330.2 μg/mL or 1.3302 mg/mL, after accounting for the dilution …show more content…
factor.
Unknown D was diluted by a factor of 1 μL UC/1001 μL dH¬¬2O and had an absorbance of .060A. The solution had light blue coloration. The dilution was decreased to 1 μL UC/501 μL dH2O and had an absorbance of 0.140A (table 2). The resulting solution had a slightly darker blue coloration, but was still a light blue. The absorbance was taken two more times at this dilution resulting in an average absorbance of 0.125A (table 2). The concentration of Unknown C was determined to be 3.515 μg/mL using the formula, which was attained from the standard curve. The correct concentration of Unknown D was found to be 1760.9 μg/mL or 1.7609 mg/mL, after accounting for the dilution factor.
Discussion:
The Bradford assay showed supportive results to indicate that it is an effective method of determining total protein concentration in solution. A standard curve was able to be produced using sample solutions of bovine serum albumin in varying concentrations. The samples ranged from a concentration of 2 μg/mL to 10 μg/mL and absorbances were taken at a 595 nm. Using the absorbance readings from these standard samples produced a standard curve with a range of 0.047 A to 0.317 A. From the standard curve a linear least- square regression line was formed, resulting in a standard curve formula, y = 0.0318x + 0.0129, with an R2 value of .9479 (figure1). The R2 value was low because the absorbance taken for 2 μg/mL and 4 μg/mL were slightly outside of the trendline.
The standard curve was then used to determine a dilution factor for the two unknown protein solutions that were going to be used.
Sample C was first diluted by a factor of 1/1000 and had an absorbance of .075A. The reading was within the standard curve range, however it was on the cusp of the range, so to be certain the absorbance would not fall from range when the experiment was replicated the dilution factor was decreased to 2/1000 or 1/500. At this concentration the sample had an absorbance of .089A. This dilution factor was used and repeated in triplicates resulting in an average absorbance of 0.097A. Using this absorbance we were able to predict the final concentration of unknown C to be approximately 1.3302 mg/mL. The same dilution was used for Unknown D and produced an average absorbance of .125A, resulting in a final concentration of 1.7609
mg/mL.
Through the experiment there were some factors that may have led to a lower than expected R2 value. There could have been an error with equipment readings. Previous experiments had seen some inconsistency with the absorbance spectrometers and even though the same instrument was used throughout the experiment, slight variation could have occurred. Human error may have also been a factor through lightly different incubation times, slightly less or more dye volume being added, or pipet issues. Some of the pipets that were used had been over dialed which can cause improper calibration.
These results were still significant in support of the Bradford assay can be used to determine the total protein concentration. The first goal of the experiment was successfully completed, resulting in a standard curve. The second goal of the experiment; determining unknown protein concentration, was supported with the results, but not conclusive because of possible errors with instrumentation or technique.
Bradford Assay: Creating a Standard Curve and Determining Unknown Protein Concentrations
Introduction:
In biochemistry the ability to determine the quantity of a certain substance in a solution is extremely important. Often protein concentration within a biological organism is essential in determining the function or condition of that organism. If the protein concentration in an environment is increased or decreased it can have detrimental side effects. If there is a decrease in hemoglobin (a protein found within blood), oxygen will not be able to be transported throughout the body. This can lead to cell death where the oxygen does not reach; therefore knowing that the body has the correct concentration of hemoglobin is important. …show more content…
The protein concentration can be measured using various methods such as Mass Spectrometry, Western blots, and colorimetric assays. Colorimetric assays determine the solute concentration of a solution based on the depth of color of the solution. For example a solution that has a deeper color will result in a higher concentration of solute. Since colorimetric assays rely on color concentration, a dye must be added to the solution so the absorbance can be taken. There have been many adaptations of colorimetric assays that test for various substances, such as the Bradford assay for total protein.
The Bradford assay for total protein is a colorimetric assay that measures the total concentration of protein in a solution. Since the Bradford assay is a colorimetric assay a dye must be used to color the solution for light to absorb in the substance. The dye used in a Bradford Assay is known as Coomassie Blue G-250, which will bind to protein when in an acidic solution1. The absorbance of samples with known concentrations can be taken and, using Beer-Lamberts Law, a standard curve can be produced. The standard curve will produce an equation in the form of, y = mx + b; which can be used to determine the concentration of an unknown sample. However, the Bradford assay is only effective between a concentration range of 1.2 μg/mL to 10 μg/mL. Therefore the samples must be made to be within this range.
The experiment aims to construct a standard curve using the Bradford Assay and varying concentrations of a known protein, bovine serum albumin (BSA). The absorbance will be taken at 595nm, due to the appropriate absorbance range for BSA. Using the standard curve produced by BSA, the concentrations of two unknown proteins will be determined.
Methods:
Preparation of Standard Curve:
A stock solution of bovine serum albumin (BSA) with a concentration of 1 mg/mL was diluted down to five solutions (in triplicate) with concentrations of 0 μg/mL, 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL, and 10 μg/mL. An equivalent volume (in ratio to the entire solution) of dye, Coomassie Blue G-250, was then added to the solution. The solutions containing the dye were then incubated for ten minutes. The absorbances of these solutions were then taken using a Perkin- Elmer Lamda Bio spectrometer, at an absorbance of 595 nm. A graph that plotted average absorbance verses concentration was created and a least-square linear regression line was obtained to determine the standard curve formula; in form of y = mx + b, when y is absorbance, m is slope, b is y-intercept, and x is concentration.
Determination of Unknown Protein:
Two unknown protein samples were selected at random, Unknown D (UD) and Unknown C (UC). The unknowns were then diluted down to various, random concentrations. The diluted solutions were combined with equivalent volumes of Coomassie Blue G-250 and left to incubate for ten minutes. The absorbances were then taken at 595 nm. The samples were diluted until the measured absorbance fell within range of the standard curve.
Results:
Five sample solutions of BSA with concentrations of 2 μg/mL, 4 μg/mL, 6 μg/mL, 8 μg/mL, 10 μg/mL were made, in triplicate, using 1 mg/mL BSA (table 1).
Calculations are as followed:
Sample (Tube) A was prepared with pure dH2O since the concentration of protein was 0 μg/mL. Upon addition of Coomassie Blue G-250 and the completion of the incubation period it was noticeable that solutions of higher concentration had a deeper color blue opposed to those with a lower concentration. The absorbance range for the standard curve (figure 1) was determined to be between 0.047A and 0.317A (table 1). This was determined to be the range in which the unknown absorbance had to fall.
Unknown C was diluted by a factor of 1 μL UC/1001 μL dH¬¬2O and had an absorbance of .0754A. The resulting solution had a light blue coloration. The dilution was decreased to 1 μL UC/501 μL dH2O and had an absorbance of 0.089A (table 2). This solution had a slightly darker blue coloration, however it was still a light blue in color. The absorbance was taken two more times at this dilution resulting in an average absorbance of 0.097A (table 2). The concentration of Unknown C was determined to be 2.655 μg/mL using the formula, which was attained from the standard curve. The correct concentration of unknown C was found to be 1330.2 μg/mL or 1.3302 mg/mL, after accounting for the dilution …show more content…
factor.
Unknown D was diluted by a factor of 1 μL UC/1001 μL dH¬¬2O and had an absorbance of .060A. The solution had light blue coloration. The dilution was decreased to 1 μL UC/501 μL dH2O and had an absorbance of 0.140A (table 2). The resulting solution had a slightly darker blue coloration, but was still a light blue. The absorbance was taken two more times at this dilution resulting in an average absorbance of 0.125A (table 2). The concentration of Unknown C was determined to be 3.515 μg/mL using the formula, which was attained from the standard curve. The correct concentration of Unknown D was found to be 1760.9 μg/mL or 1.7609 mg/mL, after accounting for the dilution factor.
Discussion:
The Bradford assay showed supportive results to indicate that it is an effective method of determining total protein concentration in solution. A standard curve was able to be produced using sample solutions of bovine serum albumin in varying concentrations. The samples ranged from a concentration of 2 μg/mL to 10 μg/mL and absorbances were taken at a 595 nm. Using the absorbance readings from these standard samples produced a standard curve with a range of 0.047 A to 0.317 A. From the standard curve a linear least- square regression line was formed, resulting in a standard curve formula, y = 0.0318x + 0.0129, with an R2 value of .9479 (figure1). The R2 value was low because the absorbance taken for 2 μg/mL and 4 μg/mL were slightly outside of the trendline.
The standard curve was then used to determine a dilution factor for the two unknown protein solutions that were going to be used.
Sample C was first diluted by a factor of 1/1000 and had an absorbance of .075A. The reading was within the standard curve range, however it was on the cusp of the range, so to be certain the absorbance would not fall from range when the experiment was replicated the dilution factor was decreased to 2/1000 or 1/500. At this concentration the sample had an absorbance of .089A. This dilution factor was used and repeated in triplicates resulting in an average absorbance of 0.097A. Using this absorbance we were able to predict the final concentration of unknown C to be approximately 1.3302 mg/mL. The same dilution was used for Unknown D and produced an average absorbance of .125A, resulting in a final concentration of 1.7609
mg/mL.
Through the experiment there were some factors that may have led to a lower than expected R2 value. There could have been an error with equipment readings. Previous experiments had seen some inconsistency with the absorbance spectrometers and even though the same instrument was used throughout the experiment, slight variation could have occurred. Human error may have also been a factor through lightly different incubation times, slightly less or more dye volume being added, or pipet issues. Some of the pipets that were used had been over dialed which can cause improper calibration.
These results were still significant in support of the Bradford assay can be used to determine the total protein concentration. The first goal of the experiment was successfully completed, resulting in a standard curve. The second goal of the experiment; determining unknown protein concentration, was supported with the results, but not conclusive because of possible errors with instrumentation or technique.