Protein Expression and Purification Series - Bio-Rad

Protein Expression and Purification Series
Focus Questions: SDS-PAGE Electrophoresis
1.
What are four ways to quantify the amount of protein in a sample?
1, Absorbance at 280 nm using a spectrophotometer; 2, Bradford or Lowry protein assay; 3, ELISA;
4, SDS-PAGE or polyacrylamide gel analysis.
2. What does primary (1°),
secondary (2°), tertiary (3°), and quaternary (4°) protein structure
refer to?
Primary protein structure refers to the amino acid sequence of the protein. Secondary protein structure
refers to the hydrogen bonds that link the amino acids and cause pleating or spiraling. Tertiary
structure refers to the 3-D structure of the protein as it is folded up. Quaternary structure refers to the
protein complex—more than one amino acid chain, or subunit, joined to make a functional protein.
3. Describe SDS-PAGE analysis. Why would you use SDS-PAGE to analyze your samples?
Sodium dodecyl sulfate polyacrylamide gel electrophoresis is a technique used to separate a mixture
of proteins based solely on protein size with the use of a gel medium and an electric field. The
relative quantities of different proteins as well as the sizes of those proteins can be visualized using
SDS-PAGE. Therefore, for protein expression and purification, because the size of the recombinant
protein produced is known, SDS-PAGE can be used to visualize if the protein is expressed, soluble or
insoluble, and how well it was purified.
4. What are BME and DTT and what do they do to a protein?
BME, β-mercaptoethanol, and DTT, dithiothreitol, are reducing agents that disrupt a protein’s disulfide
bonds that help stabilize the structure of some proteins.
5. Describe the components and function of each component of Laemmli sample buffer.
Laemmli sample buffer is a commonly used SDS-PAGE sample buffer. It consists of Tris to maintain pH
conducive to electrophoresis, glycerol to weigh the sample, SDS to equalize protein charge, potentially
a reducing agent to break protein disulfide bonds, and bromophenol blue, which gives the sample
color.
Appendix H: Focus Question Answers
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APPENDIX H
FOCUS QUESTIONS
INSTRUCTORS GUIDE