To install StudyMoose App tap and then “Add to Home Screen”
Save to my list
Remove from my list
The Bradford protein assay is a quantitative assay that is used to calculate the presence and concentration of a protein in a solution by being dependent on the amino acid composition of the proteins measured. An assay is needed to detect the concentration of the protein in a cell when the cell is put under different stress conditions. Coomassie brilliant blue is the dye used to stain the proteins that will be worked with in the experiment. In an acidic cationic state the coomassie dye has a reddish brown color to it, and in a basic anionic state it is a blue color.
More coloration means there is more protein that is present in the sample, which is detected with a spectrophotometer. The dye will have a phase shift from reddish brown to blue once it is under stable conditions, being observed at 595 nm wavelength of light.
Therefore, the amount of the concentration of the protein present will be directly proportional to the color intensity that will be observed.
Verified writer
Proficient in: Chemistry
5 (339)
“ KarrieWrites did such a phenomenal job on this assignment! He completed it prior to its deadline and was thorough and informative. ”
As for the chemical mechanism, the reddish brown dye has a lone pair of electrons that is donated to an ionizable group of amino acids in the native protein, resulting in the native structure of the protein to become distorted and unstable. This results in most of the hydrophobic residues of the protein to become exposed to the outside due to the native protein disruption. Now that both the dye and protein are unstable, they form an ionic bond between themselves, turning the dye from reddish brown to blue.
Utilizing the Bradford Assay is an efficient way to determine the concentration in an unknown sample.
To acquire a standard curve, BSA samples were used. To be used in the BSA standards reactions, cuvettes were labeled 1-7, and an eighth blank cuvette was labeled as well. One milliliter of dye reagent was pipetted into each of the eight cuvettes. In order to prepare the blank in the eight cuvette, 20 μL of dH2O was pipetted into the blank, and 20 μL of each BSA standard was pipetted into cuvettes 1-7 so the samples were prepared for the calibration curve. Each sample was mixed by being covered with Parafilm and carefully inverting multiple times, making sure not to spill the contents within. After waiting 5 minutes, the spectrophotometer set at 595 nm was used to read the absorbance of each cuvette, making sure to reset the blank each time. This was done by putting cuvette eight in the spectrophotometer and pressing the blank to set an absorbance of 0.000. The absorbance data in samples 1-7 were then observed and collected.
Measuring the protein concentration of the two milk samples was next. A blank was prepared by pipetting 20 μL dH2O into the blank cuvette and 1 mL of the Bradford reagent. Independent cuvettes were labeled with the identity of the milk sample being analyzed, and then were pipetted with 20 μL of each milk sample. Then 1 mL of dye reagent was added to each cuvette. Again, the samples were mixed by being covered with Parafilm and inverted multiple times very carefully. Five minutes was then waited out. The spectrophotometer was set to 595 nm, the cuvette was placed with the blank, and the blank button was pressed to set the absorbance to 0.000.
Each milk sample's absorbance was read and the data was recorded. The absorbance of the sample must be below 1 unit in order to proceed, if it is greater than 1 unit, the solution in the cuvette was discarded and started over using diluted solutions of the original milk samples. This was done by properly preparing and labeling the solution in new Eppendorf tubes, and then completing the dilution using a buffer.
Table: Milk Samples Used
| Sample Name | Data in the Nutrition Fact Panel - Grams of protein |
|---|---|
| Sample 1 | 1.0 g |
| Sample 2 | 8.0 g |
Table 1. Absorbance Values for BSA Standards (Calibration Graph):
| Protein Concentration (mg/L) | Absorbance (Trial 1) | Absorbance (Trial 2) | Absorbance (Trial 3) | Mean Absorbance |
|---|---|---|---|---|
| 0.125 | 0.296 | 0.298 | 0.301 | 0.298 |
| 0.25 | 0.361 | 0.362 | 0.363 | 0.362 |
| 0.50 | 0.536 | 0.525 | 0.540 | 0.534 |
| 0.75 | 0.589 | 0.604 | 0.605 | 0.599 |
| 1.00 | 0.691 | 0.697 | 0.707 | 0.698 |
| 1.50 | 0.821 | 0.829 | 0.836 | 0.829 |
| 2.00 | 0.933 | 0.929 | 0.938 | 0.933 |
*To calculate the mean, add up all three trials that correlate with the protein concentration and divide that number by three. For example, for the protein concentration of 0.125, add 0.296, 0.298, and 0.301 together to get 0.895, and then divide by three to get 0.298.*
Table 2. Protein Concentration in the Two Milk Samples
| Commercial Sample | Absorbance (Trial 1) | Absorbance (Trial 2) | Absorbance (Trial 3) | Mean Absorbance | Dilution Factor |
|---|---|---|---|---|---|
| 1 | 0.536 | 0.537 | 0.556 | 0.543 | 1 |
| 2 | 0.577 | 0.633 | 0.574 | 0.595 | 4 |
*To calculator the mean, add up all three trials that correlate with the commercial sample and divide that number by three. For example, for the commercial sample of sample 1, add 0.536, 0.537, and 0.556 together to get 1.629, and then divide by three to get 0.543.*
The equation of the linear regression on the plot is y = 0.3344x + 0.3149, with the slope being 0.3344 and the intercept being 0.3149. The concentration of the unknown protein (x) is calculated by inputting the absorbance sample (y), and then multiplying by the dilution factor used in the experiment. In commercial sample 1, trial 1, the absorbance was 0.536 with a dilution factor of 1. Calculations are shown below:
y = 0.3344x + 0.3149
0.536 = 0.3344x + 0.3149
0.2211 = 0.3344x
x = (0.6611)(1)
x = 0.661 g/L
In commercial sample 2, trial 1, the absorbance was 0.577 with a dilution factor of 4.
Calculations are shown below:
y = 0.3344x + 0.3149
0.577 = 0.3344x + 0.3149
0.2621 = 0.3344x
x = (0.7838)(4)
x = 3.135 g/L
The Bradford Assay is a fast and cost effective experiment that focuses on determining the concentration of protein in different samples of milk. Coomassie blue dye was used to bind the specific amino acids because the dye only binds to amino acids with aromatic or basic side chains.The reddish brown to blue color change of the mixture changes the absorbance of the different samples. Using the Bovine serum albumin (BSA) standard, which are proteins used as a standard protein concentration, allowed for the making of the calibration curve graph. The specific protein concentration of each sample was determined by the absorbances due to the BSA standards.
Milk sample 2 had the highest protein concentration, being 3.346 g/L, whereas milk sample 1 had the lowest protein concentration, being 0.682 g/L. It was difficult to have any observation as to which milk sample would have the highest protein concentration, considering what type of milk both samples were was unknown. If one of the samples was known to be sourced from an animal as compared to a nut milk, it could be assumed that the animal milk sample would have a higher protein concentration. As for percent error, the percent error for Milk.
Sample 1 was 84% and the percent error for Milk Sample 2 was 90%. Calculations for Milk Sample 1 are shown below.
error × 100% = Listed Concentration
error × 100% = 4.17 L
|0.682 −4.17 |L
error 84%% =
Both of these percent errors show that there was a large error during the experiment. If the percent error was small, that would mean that you are near the value expected. There were a few different steps that could have resulted in a source of error. One example would be how the milk volumes were measured in microliters, making it more tedious to ensure that all of the pipetted milk was correctly put into the buffers to dilute the samples.
Another possible source of error would be that although the experiment included triplicate data, it could have been challenging to read the absorbances at the five minute mark, as indicated in the lab manual. This could have affected the absorbance because the dye was not completely bonded to the proteins in each trial compared to if the solutions stayed in the spectrophotometer longer. Again, the Bradford Assay is a fast and less expensive experiment that determines the different concentration of proteins, which is relevant to all different fields of sciences. Other than milk, protein concentration is important in many different types of molecules, kinetics, drugs, and so forth. Performing this experiment highlights the importance of being able to acquire proper protein concentrations during a laboratory experiment.
Protein Quantification in Milk Using the Bradford Assay. (2024, Feb 17). Retrieved from https://studymoose.com/document/protein-quantification-in-milk-using-the-bradford-assay
👋 Hi! I’m your smart assistant Amy!
Don’t know where to start? Type your requirements and I’ll connect you to an academic expert within 3 minutes.
get help with your assignment
