User:Michael S. Bible/Notebook/571/2014/10/01

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Analysis of Dialysis Begun Yesterday

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
    • Don't forget to run a blank with just Bradford & buffer
    • Don't forget to run your undialyzed Lysozyme stock

Procedure taken from Dr. Fox

Data

Image:Bradford_Analysis_of_All_Post_Dialysis_Solutions.png

Figure 1 shows the corrected UV-Vis Bradford Spectra for all Post-Dialysis Solutions.

Image:Bradford_analysis_of_post_dialysis_solutions_from_non_protein_side_of_dialysis_chamber.png

Figure 2 shows the corrected UV-Vis Bradford Spectra for the post-dialysis solutions from the non-protein side of the dialysis chamber. From the negative absorbance values, it is clear that there is no protein present. This is expected since the dialysis tubing used had a molecular weight cut-off of 3500 g/mol.

Image:Bradford_Analysis_of_post_dialysis_solutions_from_protein_side_of_dialysis_chamber.png

Figure 3 shows the corrected UV-Vis Bradford Spectra for the post-dialysis solutions from the protein side of the dialysis chamber. It is clear from the spectra that there is still a lot of protein in each of the chambers, and the post-dialysis concentration of Lysozyme in solution almost perfectly matches the concentration of Lysozyme in the stock solution.

Image:Lysozyme_Concentrations_via_Bradford_Post_dialysis.png

Figure 4 shows the calculations done to determine the concentration of Lysozyme in each solution. From these calculations, it would seem that the actual concentration of stock Lysozyme solution is .28 g/L rather than .5 g/L as believed. This is because the stock solution is diluted by a factor of 50 when it is mixed with Bradford reagent and buffer.




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