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…
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…
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
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…
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…
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