User:Roberta Diaz Jimenez/Notebook/CHEM 471 Experimental Lab/2015/09/09

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Objective

Today's lab has two major goals:

  1. To determine the molar absorptivity of lysozyme which will later allow us to quantify protein concentration using the Bradford assay.
  2. Using different protein concentrations in solution, determine how fluorescence intensity is affected.

Procedure

We followed the protocol Dr. Hartings detailed in his notebook.

  1. Mass of lysozyme: 0.00732g
  2. Stock solution concentration: 51.2µM

We made five solutions each with different concentrations ranging from 0 to 15µM; we added a specific amount of solution to a 10mL volumetric flask and filled the rest with water:

  1. 15µM solution: add 2.93mL of our stock solution
  2. 7.5µM solution: add 5mL of 15µM solution
  3. 5.25µM solution: add 7mL of 7.5µM solution
  4. 2.625µM solution: add 5mL of 5.25µM solution
  5. 0.7875µM solution: add 3mL of 2.625µM solution

For the part concerning the protease, since we are group 3, we used Trypsin.

  1. Mass of trypsin used: 0.00122g
  2. Mass of eppendorf tube: 1.0325g
  3. Concentration of Trypsin in 1mL of water: 52.36µM

Data

RAMG2 Lysozyme Fluorescence Graphsept9.png

Lysozyme Molar Absorbtivity Graph.RAM.png

  • Lysozyme Molar Absorbtivity Coefficient: 37700 M-1 cm-1

Matt Hartings For your A vs concentration data show your data as points and not a curve. What is your calculated molar absorptivity in M-1 cm-1?