Can someone help me calculate protein concentration using the Bradford Assay?
If you could show me how to do tube 2, that'd be great.
Tube 1: 0 ul Protein Stock Solution, 800 ul Water, 0 ug Protein, Absorbance @595nm = 0
Tube 2: 10 ul Protein Stock Solution, 790 ul Water, 1.4 ug Protein, Absorbance @595nm = .068
Tube 3: 20 ul Protein Stock Solution, 780 ul Water, 2.8 ug Protein, Absorbance @595nm = .144
Tube 4: 40 ul Protein Stock Solution, 760 ul Water, 5.6 ug Protein, Absorbance @595nm = .256
Tube 5: 60 ul Protein Stock Solution, 740 ul Water, 8.4 ug Protein, Absorbance @595nm = .361
Tube 6: 80 ul Protein Stock Solution, 720 ul Water, 11.2 ug Protein, Absorbance @595nm = .464
I have to determine the protein concentration and plot it against the absorbance.
To calculate the protein concentration using the Bradford Assay, you will need to create a standard curve by plotting the absorbance values against known protein concentrations. Then, using the equation of the best-fit line, you can determine the protein concentration for each tube. Here's how you can do it for Tube 2:
Step 1: Create a standard curve using the absorbance values and known protein concentrations for Tubes 3 to 6.
Tube 3: 2.8 ug protein, Absorbance = 0.144
Tube 4: 5.6 ug protein, Absorbance = 0.256
Tube 5: 8.4 ug protein, Absorbance = 0.361
Tube 6: 11.2 ug protein, Absorbance = 0.464
Step 2: Plot the known protein concentrations (x-axis) against the corresponding absorbance values (y-axis).
Step 3: Find the equation of the line of best fit using a suitable regression analysis, such as linear regression. This equation will be used to determine the protein concentration for Tube 2.
Step 4: Using the absorbance value for Tube 2 (0.068), substitute it into the equation of the line of best fit to calculate the estimated protein concentration.
Note: It is important to have a blank reference measurement (Tube 1) to account for any background absorbance.
Once you have the protein concentration for Tube 2, you can proceed to plot it on the graph along with the other known concentrations.
To determine the protein concentration using the Bradford Assay, we need to perform a calibration curve using known protein concentrations. This curve will allow us to correlate the absorbance at 595nm with the protein concentration.
Here's how you can calculate the protein concentration for Tube 2:
1. Prepare a series of protein standard solutions with known concentrations, for example, 0.2, 0.4, 0.6, 0.8, and 1.0 mg/mL. These solutions will help you construct the calibration curve.
2. Measure the absorbance of each standard solution at 595nm using a spectrophotometer. Make sure you use the same cuvette or tube that you used for your sample tubes.
3. Plot a graph with the protein concentration (mg/mL) on the x-axis and the corresponding absorbance values on the y-axis.
4. Determine the equation of the calibration curve by fitting a linear regression line to the plot. You can use statistical software or spreadsheet programs like Excel to do this. The equation will be in the form y = mx + b, where y is the absorbance and x is the protein concentration.
5. Once you have the equation of the calibration curve, you can use it to calculate the protein concentration of Tube 2.
Let's assume the equation of the calibration curve is: y = 0.601x + 0.026
In Tube 2, the absorbance at 595nm is 0.068.
Using the formula, we can substitute the absorbance value into the equation to solve for x (protein concentration):
0.068 = 0.601x + 0.026
Rearranging the equation:
0.601x = 0.068 - 0.026
0.601x = 0.042
x = 0.042 / 0.601
x = 0.07 mg/mL
Therefore, the protein concentration in Tube 2 is approximately 0.07 mg/mL.
Repeat this process for the other sample tubes by substituting their respective absorbance values into the equation of the calibration curve to determine their protein concentrations.