Absorbance of Light vs. Concentration of Colored Solution Introduction: Performing this lab showed the importance of light in virtual drug screening because light can be used in a variety of ways to obtain different kinds of information in research. Light can be used to determine the concentration of DNA or protein in solution, tag different objects such as cells, protein structures, or bacteria in order to determine whether there is a large amount of a certain substance, and to determine the size of different particles. One main use for light in virtual drug screening is high throughput screening, which allows the researcher to do multiple numbers of tests on substances and quickly identify them. For example, a researcher can use light scattering to screen for ligands by measuring the amount of light scattered. The scattered light can be used to determine if there is an aggregation of proteins and how stable those proteins are, which informs researchers whether there is a ligand binding to the protein or not1. However, the point of this lab was to show how light can be used to determine the concentrations of either DNA or proteins in solution, which is a very important part of virtual drug screening. This lab introduced how a spectrophotometer can be used to determine the absorbance of different concentrations of a certain solution, and then how to use the information gathered and Beer‟s Law to calculate the concentration of an unknown solution. Beer‟s Law relates the length which light has to travel through the solution, the concentration of the solution, and the molar absorptivity with the absorbance of that solution. Beer‟s Law states that “there is a logarithmic dependence between the transmission of light that shines through a material and the density of the material as well as the length of the material that the light is traveling through”2. The logarithmic relationship is important to this lab because at high concentrations, absorbance is no longer directly related to the concentration of the solution and at around the same time the detection limit of the spectrometer is reached. Therefore in order to obtain a fair amount of accurate readings, dilutions must be made so that the concentration of the solution is below the spectrophotometer threshold. Materials and Methods: This lab started with a series of dilutions of 1mL of a 5mM stock solution provided by a mentor. The series of dilutions began with two 10 fold dilutions and then six 2 fold dilutions with each 10 fold dilution consisting of 1mL of the previous dilution and 10mL of deionized water and each 2 fold dilution consisting of 5mL of the previous dilution and 5mL of deionized water. The materials needed for the dilutions include a 10mL pipette and an electronic pipette boy to add the deionized water, a 5mL pipette and the same electronic pipette boy to transfer the appropriate amount of the previous dilution to the new dilution, and several test tubes to hold each dilution. The second part of this lab was to take absorbance readings of the multiple dilutions using the spectrophotometer. First the wavelength at which the substance absorbs light maximally must be determined. To start, the spectrophotometer must be calibrated and this was Senisterra, G.A.; Markin, E.; Yamazaki, K.; Hui, R.; Vedadi, M.; Awrey, D.E., Screening for Ligands Using a Generic and High-Throughput Light-Scattering-Based Assay. J Biomol Screen 2006, 11, (8), 940-948. 2 Pace, C.N.; Vajdos, F.; Fee, L.; Grimsley, G.; Gray, T., How to measure and predict the molar absorption coefficient of a protein. Protein Science 1995, 4, (11), 2411-2423. 1
done by using a 1mL pipettor to transfer 1mL of deionized water into a plastic cuvette. The plastic cuvette was then placed into the spectrophotometer so that the light passed through 1cm of the liquid. Then in order to calibrate, the „Calibrate‟ option on the spectrophotometer must be selected so that the machine could be provided with a...
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