User:Andrew W Long/Notebook/CHEM-671 Research/2015/10/07

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Pierce Quantitative Fluorometric Peptide Assay of the Digestion of Lysozyme by Proteinase-K

Mass lysozyme used: 0.00124g (1.24mg)

Detailed procedure can be found here

A stock solution of lysozyme was made by adding 1.24mg lysozyme to 1mL of 50mM pH 8 phosphate buffer. (1.24 mg) / (1 mL) = 1.24 mg/mL lysozyme.P he concentration of proteinase-K was determined in the following way: (0.00085g)*(1mol/28,900g)*(1/0.001L)= 0.0000294118 M proteinase-K. The amount of proteinase-K solution needed for 1mL with 1μM concentration was determined in the following way: M1*V1 = M2*V2 => (29.4118 μM)*(V1) = (1 μM)*(1 mL) => V1 = 33.99 μL Proteinase K. The amount of lysozyme stock needed to make the final solution 1 mL: (1mL total)-( 33.99 uL proteinase-K solution) = 966.01 uL lysozyme. 966uL lysozyme and 34uL proteinase-K were combined and allowed to mix and incubate. Standards were prepared by adding 75uL each standard to 75uL of water. Blanks were made by adding 75uL of each blank to 75uL of water. After incubation for the allotted times, 20uL of sample or blank, 140uL of assay buffer, and 40uL of assay reagent were mixed into the cuvette. The sample was scanned with an excitation wavelength of 390nm, with an emission from 400-650nm. The results from the assay are shown below, followed by the calibration curve for the fluorescence assay:

2015 10 7 Fluorescence of Proteinase K.png

2015 10 7 Lysozyme Fluorescence Calibration Curve of Proteinase K.png