User:Susan Schultz/Notebook/Experimental Biological Chemistry/2011/09/13
|MBP Concentration Determination||Main project page|
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Use a series of BSA standard solution to determine the linear relationship between protein concentration and absorbance.
Use the regression line to determine the concentration of an unknown sample of Maltose Binding Protein.
Prepare a starter culture for bacterial growth and protein expression.
A calibration curve was established using six standard solutions of BSA. The standard solutions of approximately 0.73, 1.46, 3.65, 5.84, 8.03, and 10.22μg/mL were prepared and then added to 200 μL of Bio-Rad protein assay. In disposable cuvettes a UV-VIS spectra was taken from 200-800nm using a Shimadzu UV-2550 UV-VIS spectrophotometer. Specifically the absorbance of each solution at 595nm was noted. A regression line was found to fit the absorbance vs. concentration data. In the future this correlation may be used to establish the concentration of MPB in a solution. This method was then tested to determine the concentration of an unknown sample of MPB. A 1/1000th dilution of the unknown sample was added to 200 μL of Bio-Rad protein assay. The measured absorbance was used with regression line established from the BSA standards to determine the concentration. A reference spectra of BSA in water was also taken using a quartz cuvette.
Figure 1. Absorbance of MBP standards
Figure 2. The absorbance of protein in water adjusted so that A→0.
Figure 3. Molar Absorbtivity v. Wavelength for protein in water
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