Week Seven – Studies on Isolated Mitochondria

Indeed, borrow all of the brains that you can for this study. Your instructor and teaching assistants are prepared to help you understand what is going on at each step of each experiment. Work with each other on the details as well. When you come in, you should already have an idea of what should happen. During the experiments, discuss your predictions with each other. Try to come up with explanations for why some results don't match predictions. Ask lots of questions. This is to be a learning experience.

Monday presentation

Prepare for a talk on preparing mitochondria from fresh liver tissue and how we study mitochondria in vitro. We will talk about paths of electrons from specific substrates, proton pumping, oxidative phosphorylation, and respiratory control. Time permitting, the talk will describe expected responses to electron transport inhibitors, uncouplers, and inhibition of ATP synthase. A web version of the slide presentation with notes is available on line.

On your own – before your laboratory session

• Prelab #6 is to be completed and submitted electronically by midnight before your lab day this week

Preparation for the polarographic studies on mitochondria

• Bring an outline of experiments to conduct and notes on what you expect to happen as you conduct the studies
• Your team 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
• Study the procedure for mitochondria isolation under "Mitochondria in vitro" (Lab guides – Mitochondria)
• You should recognize the similarities and differences between the mitochondria preparation and our previous fractionation of blood and erythrocytes
• 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
• An oxygraph training document is available as a pdf, for you to use to review the procedures
• For details on mitochondria structure and function focus on Mitochondria theory and on the remaining pages under Mitochondria in vitro
• Focus your attention on structure and function of mitochondria, including both the theoretical details and behavior of mitochondria in vitro
• You must be able to explain what is meant by respiratory control and how ATP synthase exploits the chemiosmotic gradient
• 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 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 prepared to articulate questions, particularly if you cannot explain a result.

Pre-laboratory orientation

• We will assign teams of up to six students each to work on the dissection and share tissues
• We will assign oxygraph stations for teams to share
• We will go over the importance of working as a team on the individual experiments

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

Laboratory work this week

Teams of up to six students will dissect a freshly euthanized rat to remove the liver and heart. In teams of two you will prepare isolated mitochondria from a portion of tissue. As a team of six you will operate three oxygraph chambers, conducting experiments on your isolated mitochondria and sharing the data . The team preparing heart tissue will run out of material, and will have to use liver mitochondria for the remaining experiments. Be prepared for qualitative differences between heart and liver mitochondria. Results from liver mitochondria will provide the findings for your research paper.

Expectations

• 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 efficiently without contaminating the reagents
• You are expected to make arrangements to share the data (chart records and results of the fractionation).
• 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
• There is no point in repeating an experiment that doesn't "work," without changing something
• We expect you to recognize when a result does not make sense or when a system is not responding properly
• 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
• 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.

Follow-up work

Remaining follow-up work is described on the Assignments page.