Week Five – Studies on Isolated Mitochondria

Resources

• Study the procedure for mitochondria isolation under "Mitochondria in vitro" (Lab guides – Mitochondria)
• For details on mitochondria structure and function focus on Mitochondria theory and on Mitochondria in vitro
• The oxygraph training document is available as a pdf, for you to use to review the procedures

Some expectations

You should recognize the similarities and differences between the mitochondria preparation and our previous fractionation of blood and erythrocytes.

You were to have gone through the rationale behind the study already, and you now have experience with polarography. Focus your attention on structure and function of mitochondria, including both the theoretical details and behavior of mitochondria in vitro. You must be prepared to follow a fairly fast-paced talk. You should be able to answer questions related to pathways of electron transport on specific substrates, be able to describe the sites at which protons are translocated, and be prepared to explain generation of a chemiosmotic gradient. You must be able to explain what is meant by respiratory control and how ATP synthase exploits the chemiosmotic gradient.

Recommended resources

• To complete the prelab assignment and annotated outline of experiments you will need to use the same resources that you have used to prepare for the polarographic studies
• As usual, rely on previous instructor's comments, general guidelines, examples, and specific guidelines for the research paper when you prepare your results section.
• Pages 66-71 of McMillan are particularly relevant to the writing assignment

Expectations

You should be ready to conduct a differential centrifugation procedure similar to that you conducted during the blood fractionation. From prior training you must be prepared to to conduct experiments using our polarographic systems.

You must be prepared to respond to questions from an instructor or teaching assistant with regard to experimental results and design of specific experiments. You must be prepared to articulate questions, particularly if you cannot explain a result.

Your team (see "lab work" below) should meet briefly in advance of the lab to determine how to organize the experiments, maximize your efficiency, and be prepared to duplicate experiments that return questionable results.

Expectations

You are expected to pay attention to the prelab talk, which will cover the dissection procedure and good technique for using dissecting instruments. You must be clear on the procedure for isolating mitochondria, including the reason behind each individual step and details such as which beaker to use, what medium to use, and when to top off or not top off a centrifuge tube.

You are expected to work with your team in conducting experiments, assist each other, and keep track of what the other two people are doing. We expect you to share some equipment, supplies, and reagents. You should organize yourselves so as to share the materials efficiently. You are expected to make arrangements to share the data (chart records and results of the fractionation).

We expect you to respond to questions from an instructor or teaching assistant and to articulate questions about an experiment when you cannot explain a result.

We expect you to recognize when a result does not make sense or when a system is not responding properly.

We expect you to complete the four mandatory experiments, obtaining data with which the model system can be thoroughly evaluated. We expect you to attempt the optional experiment if time permits and if the preparation is satisfactory.

Your last set of notes should be sufficiently detailed that one could prepare mitochondria and conduct the same polarographic studies, using only your notes as a guide. As always, when you finish the last experiment you should be ready to write a brief summary then turn in your materials.