User:Monika Gasiorek/Notebook/CHEM-571 2014F/2015/03/17
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March 17th, 2015
Bradford Analysis of Thermolysin Reaction Samples
250 μL of each protein sample at every time point was combined with 200 μL of Bradford reagent (1:4 Bradford:Tris/CaCl2), and 550 mL of Tris/CaCl2 buffer and run from 400 - 800 nm on the UV/Vis spectrophotometer. The following are the results with the blank subtracted out and zeroed at a wavelength of 535 nm.
Control Lysozyme Digestion
55.6 μM lysozyme stock solution was prepared from 0.08097 g of lysozyme in 100 mL of water. 0.5 mL of this stock solution was combined with 4.5 mL of water in order to produce 5 mL samples of 5.56 μM lysozyme solution.
8 of these 5 mL samples were prepared, and each of the four proteases (proteinase K, trypsin, chymotrypsin, and thermolysin) were added in the amounts specified in their respective reaction protocols to achieve final protease concentrations of 1 and 100 nM.
A 1 mL sample from each protease digestion was collected after 120 min. As stated previously, these will be used as a point of comparison when running gels in the future.
100 nM Chymotrypsin In Situ Kinetics
10.4 μL of the stock was diluted with 1.99 mL Tris/NaCl/CaCl2 buffer.