Compare And Contrast Bradford And Dc Protein Assay

Objective: The main objective for this lab was to use the Bradford and DC Protein Assay in order to determine our concentration of E. Coli BL21(DE3) lysate.

Principle of Methods: To accomplish this objective, each pair of lab partners received an E. Coli lysate sample which we had to thaw by warming it with our hands and centrifuge it. We extracted a total of 12 samples with known concentrations of bovine serum albumin and 10 samples of different amounts of lysate put into them. Using the different assays explained later in this report, we could plot a standard curve with the different amount of BSA concentrations on the x-axis as the independent variable and the absorbance measured on the y-axis as the dependent variable. The tables below
This is used because using Beer’s Law (A=bce) we know that there is a linear relationship between absorbance and concentration. As we increase the amount of lysate in our sample we should see the same increase in our measured absorbance. We can use the “best-fit” line tool on Excel to find this linear equation. We used the standard curve in this experiment to compare the increase in BSA concentration and its absorbance to our absorbance measured of the lysate to find out the unknown concentration of the lysate
This is because once the reaction reaches this point there is now less substrate for the enzyme catalyze the reaction with, thus the production of GS-DNB slows down and the absorbance increases slower as well. Therefore, you can see a plateau on the graph for our cuvette 5. Cuvette 5 is the only sample we measured the absorbance for 8 minutes, it also had a higher amount of lysate containing the enzyme. For cuvette 5 the absorbance started to level off because there was not much substrate left to catalyze the reaction, which means that the GS-DNB in the sample is no longer starting to increase as much. Since the spectrometer is measuring the amount of GS-DNB by calculating the absorbance at 340 nm, it makes sense that if GS-DNB is no longer increasing in concentration, then the absorbance will no longer increase either. Spectrometers are also known to not be as accurate past an absorbance of 1.5 and ours went up to 2.193 for cuvette 5. The only source of error that could have occurred is when my partner and I were using the stopwatch to keep track of the absorbance with the 30 second intervals. Sometimes we would forget to start it and we tried our best to estimate how many seconds behind we