& Data Analysis
Protein gel electrophoresis
Protein gel analysis
Concepts and theory
Laboratory Studies – Overview
This introduction to experimental biosciences will introduce and reinforce practical skills in the context of three different laboratory investigations. The studies were chosen on the basis of workability and their coverage of many of the fundamental skills that comprise the learning objectives of the laboratory programs in natural sciences and engineering. Although each study is independent of the others, you will build upon previously acquired knowledge and skills as you progress through the course.
Microscopy and study of freshwater invertebrates
The first hour of lab work will consist of a training session in how to use a research quality microscope. Methods for finding specimens, focusing, and adjusting contrast will be emphasized. You will practice using the microscope by examining a number of freshwater cultures. Mental exercises will help you hone your observational and quantitative skills. Although there will be no writing assignment associated with this week's work, you will need to retain the ability to find and focus on very small specimens, so that they can be examined and measured at high magnification. The following week you will have to be prepared to conduct the study on flagella regeneration.
You will also be introduced to the requirements for keeping a comprehensive laboratory notebook, and will turn in the first set of pages.
Flagella regeneration study
You will work as part of a team in order to test a hypothesis related to regulation of the assembly of flagellar microtubules in a species of protist, Chlamydomonas reinhardi. In addition to learning to pronounce the word Chlamydomonas, you will be expected to work effectively as a team member on data collection and/or in the preparation and distribution of samples. Your team will collect the data to be used for the first research paper.
The laboratory work this week, including preparation and followup work, will introduce you to bench methods and laboratory math that you will need in order to conduct the remaining studies in the course. You will prepare a protein assay reagent from scratch, prepare standards and samples for a protein assay, run the assay, prepare and interprret a standard curve, calculate fraction yields, and learn to dilute solutions given a set of specifications. Although there will be no writing assignment on this material, you will be expected to acquire and retain the skills needed with which to accomplish this kind of work. You must be prepared to learn from any mistakes and to make up any deficiencies in bench methods or the application of math skills to the lab work.
Characterization of red blood cell membrane proteins by SDS-PAGE
This study will take two weeks plus part of a third week for analysis. The objective is to use a very common laboratory method, namely SDS polyacrylamide gel electrophoresis, to separate and identify proteins that are associated with red cell (erythrocyte) membranes. During the first week you will fractionate (take apart) whole blood in order to obtain samples of membranes and other fractions of blood tissue. You will determine protein concentrations and yields for each fraction. During the second week you will prepare samples for electrophoresis, set up polyacrylamide gels, and run the samples to separate polypeptides. The third week will be dedicated in part to analyzing the gels. The entire three week study will be written up as the second research paper.
Isolation and study of mitochondria
This time you will take apart whole liver tissue for the purpose of obtaining fairly pure suspensions of mitochondria. The objective is to determine if isolated mitochondria will make a potentially reliable experimental model. Part of the previous week's laboratory work will be dedicated to learning the instrumentation for measurement of dissolved oxygen. You will measure oxygen consumption of mitochondria samples in order to estimate rates of electron transport. You will predict how switching substrates, adding ADP, and poisoning the mitochondria with various reagents should affect oxygen consumption rates. By comparing observed with predicted results you should be able to evaluate the utility of isolated liver mitochondria as a research subject. Your findings will be the subject of the third and last paper.