Problem 2
Match the terms with the descriptions. (a) Assay (b) Molecular exclusion chromatography (c) Ion-exchange chromatography (d) Affinity chromatography (e) High-pressure liquid chromatography (HPLC) (f) Isoelectric focusing (g) Sedimentation coefficient (h) Antigenic determinant (epitope) (i) Monoclonal antibodies (j) Western blotting 1\. Separating proteins on the basis of size differences 2\. Allows high resolution and rapid separation 3\. Produced by hybridoma cells 4\. An immunoassay technique preceded by gel electrophoresis 5\. A measure of the rate of movement due to centrifugal force 6\. Separating proteins on the basis of net charge 7\. Specific site recognized by an antibody 8\. Based on the fact that proteins have a \(\mathrm{pH}\) at which the net charge is zero 9\. Based on attraction to a specific chemical group or molecule 10\. A means of identifying a protein based on a unique property of the protein
Problem 4
Although many proteins precipitate at high salt concentrations, some proteins require salt to dissolve in water. Explain why some proteins require salt to dissolve.
Problem 6
(a) The octapeptide AVGWRVKS was digested with the enzyme trypsin. Would ion- exchange or molecular exclusion chromatography be most appropriate for separating the products? Explain.
Problem 7
The detergent sodium dodecyl sulfate (SDS) denatures proteins. Suggest how SDS destroys protein structure.
Problem 8
In the course of purifying an enzyme, a researcher performs a purification step that results in an increase in the total activity to a value greater than that present in the original crude extract. Explain how the amount of total activity might increase.
Problem 10
(a) Proteins treated with a sulfhydryl reagent such as \(\beta\) -mercaptoethanol and dissolved in sodium dodecyl sulfate have the same charge- to-mass ratio. Explain. (b) Under what conditions might the statement in part \(a\) be incorrect? (c) Some proteins migrate anomalously in SDS-PAGE gels. For instance, the molecular weight determined from an SDS-PAGE gel is sometimes very different from the molecular weight determined from the amino acid sequence. Suggest an explanation for this discrepancy.
Problem 13
Differentiate between polyclonal and monoclonal antibodies.
Problem 14
Explain how immunoprecipitation can be used to purify proteins.
Problem 15
What is an ELISA, and how is it used?
Problem 16
Describe western blotting.
