Determining the concentrations of proteins is necessary for a variety of different reasons. For example, protein concentration must be known in order for proteins to be used in enzyme reactions, as the correct stoichiometry must be known. Another example is protein purification; during protein purification the quantity of protein present must be known to determine the yield1. The quantification of proteins is determined by methods known as protein assays. There are different protein assays used to determine the concentration of proteins. Three different types of protein assays used by biochemists include the Bicinchoninic Acid (BCA) protein assay, Modified Lowry protein assay, and the o-Phthalaldehyde (OPA) Fluorescent Protein …show more content…
First 1000 µL of the BSA (0.1 mg/ml BSA) protein standard was added to a microcentrifuge tube. 0 µL of the protein standard was added to Tubes 1a and 1b, 20 µL was added to tubes 2a and 2b, 40 µL was added to tube 3a and 3b, 60 µL to tubes 4a and 4b, 80 µL in tubes 5a and 5b, 100 µL in tubes 6a and 6b. 50 µL of the standard protein solution were added to each of the unknowns. Next various quantities of DI water were added to each tube. 1000 µL of DI water was added to tubes 1a and 1b, 980 µL was added to tubes 2a and 2b, 960 µL was added to tubes 3a and 3b, 940 µL was added to tubes 4a and 4b, 920 µL was added to tubes 5a and 5b, and 900 µL was added to tubes 6a and 6b. 950 µL of DI water was added to each of the unknown tubes.
The last step involves the addition of dye to the tubes and the Bradford assay experiment. 200 µL of Bradford dye were added to each of the tubes contacting the standard as well as to the tubes contacting the unknown. Next 200 µL of each standard and the unknown were transferred to a well in the microtiter plate. Next, the wavelength was set to 595 nm and the temperature was set to 25 °C, and the A595 of each sample was measured using the BioRad microtiter plate reader. Lastly, the spectral scans were obtained through the BioRad microtiter with a wavelength range of 340 to 800