METHODS IN BIOCHEMISTRY AND CELL BIOLOGY
BMB-310
It does not reflect full course policy or practice, and it does not take precedence over syllabi handed out in class.
Course Purpose and Goals
To a large extent, the study of the cell and its biochemical functions can be seen as the study of proteins. Every regulated function of a cell requires the presence of at least one protein in a role such as catalyst, processor of information, or component of a complex structure. A scientist studying a cell that contains thousands of different types of proteins must have tools to answer a wide range of protein-related questions. For example . . .
How much protein does a particular sample contain? How can I identify which of the several proteins in a complex mixture is the one I'm looking for? How can I separate my protein of interest from all the others that are present? What are the basic physical properties of my protein? If my protein is an enzyme, how can I quantify the function it performs? How can I find the most likely identification and the most probable functions of a protein about which I may know only its amino acid sequence?
One goal of this course is to give you both practical experience and a theoretical background in some of the important current research methods needed to answer fundamental questions like these - and, additionally, to give you experience working with two important types of proteins, which are essential to many investigations in BMB: enzymes and antibodies.
A further and equally important goal of the course is for you to improve your ability to reason your way through experimental situations in general. Accordingly, a great deal of emphasis will be placed in the course upon your ability to plan research procedures, to carry them out correctly and efficiently, and to extract accurate and useful information from the results. These skills are amongst the most valuable that you can acquire because they can be exported to situations well beyond your experiences in just this course.
BMB 310 is a required upper-level course for students majoring in Biochemistry and Molecular Biology (BMB). The course meets for four hours a week: one hour of lecture and 3 hours of lab. Four hours of academic credit are earned. Students who are not majoring in BMB can use credit in BMB-310 to fulfill laboratory credit to accompany both Biochemistry (CHEM-414) and Cell Biology (BIOL-307).
The lecture component of BMB 310 deals with "practical bioinformatics" - meaning, roughly, the use of basic online tools for the identification and predictive analysis of proteins, based on knowledge of amino acid sequence. The laboratory component of the course deals with the theory and practice of several important "bench" techniques for protein isolation and analysis. Laboratory work is open-ended - in several cases the formal lab time serves mainly for demonstration of or beginning of a procedure, with completion of the work to be done independently on your own schedule.
| Meeting 1 | Generation of Antibodies: Immunization of Mice with Insect Protein Extracts |
| Meeting 2 | Spectrophotometry and Protein Quantification Sequence Formats and the UniprotKB Database |
| Meeting 3 | Enzyme Assays: Basic Principles Use of BLAST for Sequence Database Searching |
| Meeting 4 | Enzyme Assays: Kinetics and Inhibitors GenBank and Prediction of Protein Sequence from DNA Sequence |
| Meeting 5 | SDS Gel Electrophoresis Sequence Alignments |
| Meeting 6 | Hydrophobic Interaction Chromatography and Ion Exchange Chromatography Domain Databases and Domain Prediction |
| Meeting 7 | Gel Filtration and HIS-tag Affinity Chromatography Subcellular Targeting Prediction Programs |
| Meeting 8 | Prediction of Transmembrane Protein Topology |
| Meeting 9 | Enzyme-Linked Immunosorbent Assay (ELISA) Prediction of Post-translational Modification Sites |
| Meeting 10 | Transblotting (in Preparation for Immunoblotting) Visualization of Protein 3D structure using PDB and Chimera |
| Meeting 11 | Immunoblotting ("Western Blotting") Protein 3D Modeling using Modeller & Phyre2 |
| Meeting 12 | Cell Culture Analysis & Identification of Proteins by Mass Spectrometry (MS) |
| Meeting 13 | Immunofluorescence Microscopy |
| Meeting 14 | Analysis of Results of Mass-Spec Protein Fingerprinting |