Lab 9: Determination of Protein Stability using Chemical Denaturation and Intrinsic Fluorescence

Lab 9 Objectives

  • Gain hands-on experience with protein denaturation
  • Determine whether your mutation affects protein stability based on measurements of the free energy of unfolding (ΔGunfolding) of wild type and mutant HCAII

Before lab, you will complete a brief at-home experiment to study protein denaturation using egg whites. This should take no more than 60 minutes. During lab, you will work with your partner to connect that experiment to the HCAII experiment. (Note that it may help to view the associated lecture material before lab as well!) The in-lab time will be relatively short this week, since you will be doing some lab work before lab.

This is the first of three Biochem 551 labs in which you will be determining whether or not your mutation of interest has a measurable effect on the function of HCAII. Before directly measuring any functional parameters, it is important to determine whether or not your mutation caused any changes in structural stability of the protein. If, for example, a mutation destabilizes the protein to such an extent that it is completely unfolded, that mutant will not be able to catalyze any reactions. However, this would be a very different case than a mutant protein that is well-folded and stable, but catalytically dead. In order to differentiate between these possibilities, in lab 9 we will compare the protein stability of wild type and mutant protein.


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Biochemistry 551 (Online Version) Lab Manual Copyright © by Lynne PROST is licensed under a Creative Commons Attribution 4.0 International License, except where otherwise noted.

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