User:Michael S. Bible/Notebook/571/2014/10/08

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Run Bradford Analysis on Post-Dialysis Solutions

Post-dialysis solutions from all of the wells in the dialysis chamber were analyzed using UV-Vis using the following protocol:

  • Remove 20 μL of solution from each chamber (10 in all) and run Bradford analysis
    • Bradford reagent should be diluted 1:4 with water.
    • Recall, 20 μL solution + 200 μL diluted Bradford + 780 μL Tris/NaCl buffer
    • PS cuvettes, measuring 400 - 800 nm
    • Run a blank with just Bradford & buffer


Procedure taken from Dr. Fox

Run Ca2+ ISE on Dialyed Samples

  • Place the contents of each dialysis well in a scintillation vial.
  • Use the ISE probe to measure the potential of each solution.
  • Use values obtained along with the equation from the Ca2+ Calibration Curve to determine the final concentration of Ca2+ in the solutions.

Data

Ca2+ ISE Data

Ca2+ ISE Data for First Dialysis.png

Above are the final concentrations of Ca2+ in the post-dialysis solutions. The final two initial concentrations do not give good data as the final concentrations measured between the protein and ion side add to a greater concentration of ions than was originally present in the initial solution. These two data points should be disregarded and Lysozyme should be dialyzed against additional concentrations of Calcium ions in order to form a better curve later.

Bradford Data

Bradford Analysis of CaCl2 Dialyed Lysozyme.png

Above are the results of Bradford anlysis of the post-dialysis protein solutions. The data does not look good, and suggests that there is no protein present. The most likely reason for such data is that the wrong side of the dialysis chamber was tested using Bradford Analysis. The samples are still stored from this experiment, so it may be possible to re-run this UV-Vis experiment.

Begin New Dialysis Experiment (Lysozyme against HCl)

A dialysis experiment was prepared using 20000 MWCO Dialysis Tubing. Samples of .125 g/L were placed in the wells of one side of the dialysis chamber. These samples were dialyzed against solutions of CaCl2 of the following concentrations:

  • 250 μM HCl
  • 125 μM HCl
  • 62.5 μM HCl
  • 30 μM HCl
  • 16.7 μM HCl

The dialysis chamber was sealed and placed on an orbital shaker at room temperature until 10/14.

Data for HCl Dialysis

  • Data from Fluorescence analysis can be found here.