Week Six – Gel Analysis and Measurement of Dissolved Oxygen

Incompetents invariably make trouble for people other than themselves.
Larry McMurtry, 'Lonesome Dove'

Monday presentation

Prepare for two presentations. There will be a presentation on SDS-PAGE analysis and a second presentation on principles and use of a polarographic system for measuring dissolved oxygen. Time permitting, we will briefly review electron transport by mitochondria. The 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.

On your own – before your laboratory session

Write up the discussion, abstract, and introduction sections for your first research paper, due at the beginning of lab this week

Recommended resources

  • Examine the presentations on SDS-PAGE analysis and on polarography, described above
  • Continue to peruse the third part of our laboratory study (protein gel analysis) to characterize 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
  • Begin to examine the web materials on electron transport in mitochondria
  • Pre-lab #4 checks your understanding of terms and concepts related to protein gel analysis, and concepts related to polarographic study of mitochondria function
  • For the write-ups consult past instructor feedback carefully, and use the general guidelines for research papers, the annotated examples, and the writing text as needed

Some expectations before starting the work - protein analysis

  • 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

Some expectations before starting the work - mitochondria study

  • 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 a polarographic system
  • Be ready to describe the principle for calibrating a polarographic system
  • Begin examining the material on mitochondria theory and mitochondria in vitro
  • 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 (by instructor)

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

Follow-up work

  • As always, turn in your notebook pages
  • Your gel analysis should be complete by the end of the afternoon, so that you can write up the results section for the first paper, due in a few weeks
  • Prelab #5 must be submitted by the morning of your laboratory session, week 5
  • 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)
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Created by David R. Caprette (caprette@rice.edu), Rice University17 Aug 95
Updated 4 Oct 09