User:Lymperopoulos Loukas/Notebook/471 LAB/2015/09/30

Biomaterials Design Lab

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Objective To make standardized curves for determining peptide concentration using fluorescence analysis. Description Dr. Hartings left a procedure for us to use today in his lab notebook. We are using the Pierce quantitative fluorometric peptide assay. (product link here) Perform a protease digest of lysozyme Make a stock lysozyme solution (~ 1mg/mL) using phosphate buffer Make your protease stock by adding 1mL to your pre-measured protease Make a sample that has a final volume of 1mL and protease concentration of 1uM Add the appropriate volume of protease sample to a clean eppendorf tube Add a volume of lysozyme solution so that the final volume is 1mL Make a blank sample (no lysozyme) Add the appropriate volume of protease sample to a clean eppendorf tube Add a volume of buffer solution so that the final volume is 1mL Add both tubes to the 37C water bath for 1hr Make standards for analysis Standard A: 150uL of reaction sample Standard B: 75uL of Standard A and 75uL of water Standard C: 75uL of Standard B and 75uL of water Standard D: 75uL of Standard C and 75uL of water Standard E: 75uL of Standard D and 75uL of water Standard F: 75uL of Standard E and 75uL of water Standard G: 75uL of Standard F and 75uL of water Standard H: 75uL of Standard G and 75uL of water Make blanks for analysis Data Notes This area is for any observations or conclusions that you would like to note. Use categories like tags. Change the "Course" category to the one corresponding to your course. The "Miscellaneous" tag can be used for particular experiments, as instructed by your professor. Please be sure to change or delete this tag as required so that the categories remain well organized.