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Week Four – 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.
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
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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 the laboratory
next week
- 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 5 of the course
(Week 5 schedule page)
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