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 5, page 151. Question 5-1. This must be determined from the experimental data. Construct a standard curve as in Figure 5-7 (page 138) using the data from the standards, and determine the mass of your unknowns from this plot. Question 5-2. These questions may be answered by comparing the molecular mass determined by non-denaturing methods, such as gel filtration chromatography, with results obtained using a denaturing method, such as SDS-polyacrylamide gel electrophoresis. for example, if a given protein has a molecular mass of 110 kDa by gel filtration chromatography, and a single type of subunit with a mass of 55 kDa is observed in SDS-gel electrophoresis, then the protein is likely to be a homodimer of 55 kDa subunits.
		To determine whether a single type of subunit is present, it is generally necessary to purify the protein, after which SDS-gel electrophoresis may be used to indicate the number of different species present. This method will generally not resolve proteins with less than 1% difference in mass, so different subunits of nearly identical mass will not be distinguished. In this unlikely case, another method must be used. One method that could be used is simple polyacrylamide gel electrophoresis in the presence of urea. Urea will cause denaturation of the oligomer into monomers, which will migrate differently in the gel due to differences in shape and charge. Unless the subunits are exactly the same size and charge, they should be resolved on this gel. Another difficulty is encountered when a protein contains a subunit in non-stoichiometric amounts. For example, RNA polymerase from Escherichia coli contains a subunit known as the omega subunit in non-stoichiometric amounts. Such subunits may be easily overlooked because one might think that they are contaminants of the purified protein. Question 5-3. The easiest way to determine whether disulfide bonds are responsible for holding an oligomer together is to compare the protein mobility on SDS-polyacrylamide gels in the presence and absence of a reducing agent such as b-mercaptoethanol. In the presence of the reducing agent, the disulfide bonds will be reduced and the protein subunits should migrate as monomers. In the absence of the reducing agent, disulfide bonds will hold the subunits together and the protein will migrate as the oligomer.

Notes on Experiments Homework Answers Book Revisions Contact Us Authors Home Links