Title: The concentration readings of different proteins
In this lab we used the Bradford method to determine the concentration amount that is in the different samples of proteins. The three main proteins used were y-globulin, PBS and the Bradford dye. Once each sample was ready to be read, I gathered them one by one. I read the absorbance concentration for each tube. With the results there could have been some mircropipetting errors or reading error but, based on the end results, my tests came close to the expected reading.
With this lab there are three different methods that could be used. There is the biuret method which is commonly used in biology but, it is also the oldest. Next is the Lowry method which is the most sensitive method, but it is used to determine the interference of chemicals. Lastly is the Bradford method which the one we used in the lab. It is used to measure the amount of concentration in certain proteins. This method is the faster method of the three. The spectrophotometer is the machine that was used to get the concentration readings of the proteins. It measures the absorbance of a protein. The objective of this experiment was to find the concentration of an unknown protein. Once completed we were able to plot the information and create a standard curve graph.
MATERIALS AND METHODS
In this lab I obtained the protein globulin (y-globulin), phosphate buffered saline (PBS) and the Bradford protein dye from the instructor and keeping them on ice. I gathered eight microcentrifuge tubes and labeled them each. I then added the correct amount of stock solution to each minus the first tube. Then following that process adding the PBS to each of the tubes following the correct amount that needed to be dispensed. Final concentrations should be 0.0, 0.2, 0.4, 0.6, 0.8, 1.0, 1.2 and 1.4. Once completed I closed the caps tightly and make sure to mix the solutions well and placing them back into the ice bucket. After that process was completed I gathered eight cuvettes and labeled them. Next, I added 1.0 ml of the dye reagent to each of the cuvettes. Following that I added 20 microliter of the correct standard to each cuvettes spacing them one minute apart.Making sure to keep the cuvettes at room temperature so that the protein binding would take place. Once the final time reaches ten minutes, I started reading the absorbance for each one and the first one should be a blank reading 0.000. Once all of the readings were completed I plotted the numbers on a graph (standard curve) see figure 1. For the next activity I gathered two microcentrifuge tube and labeled them y-globulin and BSA. I then took 100 ml of the correct protein sample while keeping them on ice. Then I got six disposable cuvettes and labeled them G1-3 and A1-3. Then I carried out the color reactions by adding 1.0 ml dye reagent and 20 microliter of the protein sample following the 10 minutes process. Once the time was completed I read the absorbance for each one and I calculated the average reading.
By using excel, I was also able to calculate the equation for a best fit line. From that equation, I could determine the protein concentration of each of the unknown samples from its absorbance shown in table 2
Table 1: This shows the Absorbances with protein concentrations.
Construction of Standard curve for BSA
Conc (mg/ml) A595
Here is listed the absorbance readings (y axis) and the concentration (x axis) of each sample that was containing the protein. From this, I discovered a negative correlation between the concentration and its absorbance. I used this data to calculate the protein concentrations of the unknown samples.
Table 2: The protein concentrations of five possible identities of the unknown substances.
Calculation of Protein Concentration
Samples A595 Conc (mg/ml)
1 0 2.1486
2 0.627 0.427485
3 0.675 0.295725
4 0.673 0.301215
5 0.426 0.97923
6 0.697 0.235335
7 0.642 0.38631
In this experiment, the goal was to find the identities of the unknown proteins by using their concentrations from the absorbance readings. The higher the absorbance meant the higher the concentration would be. From there I gathered the concentrations and compared them to the already known samples. The resulting information showed that our reading came close to the given concentrations. The readings could have came more close to the expected reading if their had not been any interference with the handling of the material or readings on the machines.
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