User:Nicole Bonan/Notebook/Chem 571 Lab Notebook/2015/09/23
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The objective for today's lab work is to create a series of lysozyme solutions and then measure their absorbances using a Bradford assay.
We based our protocol for our Bradford Assay off of the one that Dr. Hartings outlined in his lab notebook. However, we did not follow his protocol exactly; ours is outlined below.
In the table, the first column shows the number of the cuvette. We numbered the cuvette from 1 to 12 in order of increasing concentration of lysozyme. The second column shows the concentration of lysozyme in the cuvette. We arbitrarily assigned a concentration, between 1 and 10µg/mL of lysozyme in buffer, for each of the cuvettes. The third column shows the volume of the 50µg/mL stock solution of lysozyme, which we made in step 2.2, that we needed to pipette into the cuvette in order to get the final concentration that we assigned that cuvette. The fourth column shows the volume of the Bio-Rad Protein Assay reagent that we added to each cuvette. We pipetted 600µL, or 0.600mL, into each cuvette. The fifth column shows the volume of the buffer that we added to the cuvette in order to adjust the concentration and final volume of the cuvette. The last column shows the final volume of solution in each cuvette, which was always 3mL.
In order to determine the volume of the 50µg/mL stock solution that we needed in each cuvette (column #3 in the table), we did the following calculation:
The figure above shows the raw data from the Bradford Assay. It shows the absorbance of the blank and of each lysozyme sample as a function of the wavelength of incident light. Each different colored line in the plot represents a different lysozyme sample; the concentration of the samples are listed in the legend on the right hand side of the graph. These absorbance values are not corrected for noise or the blank.
The figure above shows the corrected absorbance of each lysozyme sample in the Bradford Assay as a function of the wavelength of incident light. The absorbance values were corrected by first subtracting the absorbance value of the blank from the absorbance value of each sample for every wavelength. Then, the absorbance value at the isobestic point for each sample was subtracted from all the absorbance values for that sample. The isobestic point was at 535nm. Each different colored line in the plot represents a different lysozyme sample; the concentration of the samples are listed in the legend on the right hand side of the graph.
The figure above is the calibration curve for the Bradford Assay. It shows the absorbance of lysozyme as a function of concentration of lysozyme when the indecent light has a wavelength of 600nm. The absorbance values are taken from the corrected absorbance calculated for all of the lysozyme samples from the assay. The wavelength of incident light of 600nm was chosen for the calibration curve because this wavelength is the wavelength at which the lysozyme samples showed peak absorbance.