Isolation and Characterization of Immunoglobulin from Serum

Jun 30, 2009 by Ph.D. Carlos Acevedo Suárez

Students will develop hands-on experience in the purification and characterization of immunoglobulin from serum using biochemical and immune-based approaches. The laboratory component of the Immunology course has traditionally been driven by theoretical discussions, with limited emphasis on experimental techniques. As a result, students have a poor grasp of immune-based approaches used for research. This module provides students the unique opportunity to follow an established sequence of experimental techniques that parallels those that would be performed in a true research setting. At present, it has become possible to harness the high specificity of antibodies allowing them to become essential tools for basic and applied research as well as in the clinical setting. The module will extend over 4 lab sessions and the exercises will be performed in teams of 3-4 students. They will learn affinity chromatography, protein quantitation assays, SDS-PAGE, ELISA. Each team will isolate the IgG fraction from a sample of serum by affinity chromatography. The non-IgG fractions will also be retained for further analysis. Samples will be subjected to SDS-PAGE to characterize structural features of antibodies before and after disulfide bond reduction. All fractions collected after affinity chromatography will also be subjected to ELISA to illustrate the varying protein composition of each sample by means of specific antibody reactions against serum components. A Questionnaire on research content, skills, tools and techniques will be offered on the first day of lab and after completing the lab module. Students will be also asked to submit a report encompassing all the laboratory exercises in the module. The format of the report will be that of a research article. Given that results are expected to vary for all the teams within each laboratory section, students will be able to pool their data in order to compare and contrast the results obtained by each team, thus providing another opportunity to further enhance their critical thinking skills.

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