Fundamental Laboratory Approaches
for Biochemistry and Biotechnology

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by Alex J. Ninfa and David P. Ballou

Notes on Experiments Homework Answers Book Revisions Contact Us Authors Home Links		Chapter 4, p113. Question 4-1. In anion exchange chromatography, such as DEAE-cellulose chromatography, the desired gradient would be from high pH to low pH. At high pH, acidic side chains of the proteins will be ionized, causing the proteins to bind to the column. As the pH is lowered, these side chains will become protonated, causing elution of the proteins. Proteins will elute differently, according to how they ionize at various pH values. The converse is true for cation-exchange chromatography. At low pH, basic side chains of proteins will be positively charged, causing the proteins to bind to the column. As the pH is raised, these basic side chains will become de-protonated, and the proteins will elute. and patches of negative charges on different surfaces of the protein.
		Question 4-2. One way is to determine the conductivity of a small aliquot of the sample and then to compare this conductivity to a standard curve constructed using various known salt concentrations in the same buffer. Of course, this will only be an approximation, since the proteins in the test solution will also contribute (slightly) to the conductivity. Question 4-3. The pH will be higher near the ionizable groups of a cation-exchange resin because the resin itself is negatively charged and will attract protons. Thus, a layer of bound protons requires a diffuse layer of OH- ions, resulting in higher pH in the region of solvent accessible to the protein. This is known as the Donnan effect, as explained on page 110. Question 4-4. Yes, because on some proteins there are patches of positive