Week Six – Gel Analysis and Measurement of Dissolved Oxygen

Monday presentation

Prepare for two presentations. We will talk about SDS-PAGE analysis and the talk about principles and use of a polarographic system for measuring dissolved oxygen. Time permitting, we will briefly review electron transport by mitochondria. A presentation on SDS-PAGE analysis with notes is available for viewing on-line and can be downloaded as a pdf in grayscale format for printing. There is also an on-line presentation on polarography and a pdf for downloading/printing.

Background reading for the CPR work (second assignment) will be made available some time today.

On your own – before your laboratory session

• Write up the materials and methods to the second study, to be submitted at the beginning of your laboratory session this week
• Prelab #5 is to be completed and submitted electronically by midnight before your lab day this week
• Peruse the third part of our laboratory study (protein gel analysis) to characterize membrane proteins
  • Outline how you will conduct the gel analysis, step by step
  • Be able to describe what kinds of information can be obtained from protein gels
  • Be prepared to describe a strategy for calibrating and analyzing a protein gel
  • Be prepared to recognize gel patterns that are typical of red blood cell membrane samples
  • Be prepared to tell the difference between a low percentage and high percentage gel from the pattern of bands
  • Be prepared to describe the specific types of evidence that we will collect in order to complete our characterization of red cell membrane proteins
• Find the posted images of your gels, and print off at least two good copies of each gel on which your samples were run; if it appears that you will have trouble analyzing your own data, print off copies of a suitable gel run by another group, and prepare to acknowledge them; they need not be printed in color
• Examine the Experimental rationale behind the study that we will conduct next week (preparation and study of isolated mitochondria)
• Read the material on polarography
  • Be able to explain why we are going to learn to use a polarographic system for measurement of dissolved oxygen
  • Be able to describe the components of apolarographic system
  • Be ready to describe the principle for calibrating a polarographic system
• Begin to examine the web materials on electron transport in mitochondria
  • Be able to describe the path of electrons in the electron transport system of mitochondria, from either NADH or succinate to molecular oxygen
  • Be prepared to predict what will happen to oxygen consumption when you add a substrate or an electron transport inhibitor to isolated mitochondria

Pre-laboratory orientation

• There will be a prelaboratory orientation to the gel analysis
• We will conduct a formal training session on use of the oxygen measurement equipment

Laboratory work this week

You will analyze your gels using the gel images that you bring in and the original gel that will have been stained and destained. You should finish the entire analysis and start on the actual identifications for your discussion. The more work you complete in lab the easier the second paper will be for you. Take advantage of the fact that TAs and one or more instructors will be available to help you with the analysis.

We will conduct a tutorial session on using polarographic equipment to study mitochondria function. You will conduct a quick experiment using frozen mitochondria that were prepared earlier.

Follow-up work

• Your gel analysis should be completed by the end of the afternoon, so that you can write up the results section for the first paper, due in the laboratory next week
• Prelab #6 is to be completed and submitted electronically by midnight before your lab day nexx week
• Examine the posted article on membrane proteins (pdf format); you may wish to print it off to consult as you attempt to identify unknown bands on your gels
• Start on your preparation for week 7 of the course (Week 7 schedule page)
• Either during the lab session or in a follow up email message you will receive a team assignment for conducting the mitochondria isolation and experiments.