Week Four – Blood Fractionation

The great tragedy of Science - the slaying of a beautiful hypothesis by an ugly fact.
Thomas H. Huxley (1825 - 1895)

Monday presentation

The Monday talk will present the rationale behind our laboratory study on characterizing red blood cell membrane proteins. The talk is available on line and slides with notes are available as a pdf, formatted for grayscale printing. If necesssary please peruse the presentation well in advance of your lab day so that you have time to ask questions and resolve any misunderstandings. You must be prepared for the laboratory work, including understanding is purpose.

On your own – before your laboratory session

  • Complete and submit Pre-lab #3
  • Peruse the material under "Characterizing Erythrocyte Membrane Proteins by SDS-PAGE – Part 1"
  • Prepare an outline of work to be done so that you can work efficiently on the project
    • Prepare to apply any or all of the skills and concepts covered in the "Laboratory math" study
    • Be able to describe the overall objectives and specific objectives of this week's work
    • Be able to descibe the full rationale behind red cell fractionation
    • Be able to describe how each fraction relates to the structure of blood and red blood cells
    • Be able to describe principles of differential centrifugation
    • Be able to identify and describe the protein assay that we will use

Suggestion

Division of labor is key to a good laboratory performance this week. The work can easily be completed well before 5 pm, giving you plenty of time to finish taking notes. Here is how to divide up responsibilities effectively.

  • Both of you must be prepared to apply what you learned in the "laboratory math" session
  • The two of you must be clear about who will take responsibility for what part of the project
  • You must consult with each other as you work and enter notes on what both of you are doing as you go along
  • When you finish the last bit of laboratory work you should both be ready to write summaries and go
  • You must not copy notes directly from each other's notebook; consult with each other and take notes in your own words

Pre-laboratory orientation

  • "Walk through" the fractionation procedures, including safe use of centrifuge equipment
  • Introduction to using a re-pipet to deliver color reagent
  • Go over suggestions for division of labor and record keeping

Laboratory work this week

You will conduct fractionation of whole mammalian blood to obtain washed erythrocytes, then take the erythrocytes apart to obtain washed cell membranes. You will collect an aliquot of each fraction and determine protein concentration in each aliquot using a Bradford protein assay. You will label and store your fractions for use the following week.

Follow-up work

  • Place your sample tubes in the ice bucket designated for aliquots to be frozen for the following week
  • Turn in initialed notebook pages as you are expected to do each week
  • Remember, the CPR work is to be completed by Friday this week (19 Sept), 5 pm
  • On your own, finish determining protein concentrations for your samples if you run out of time during the laboratory session
  • Start on your preparation for the fifth week of the course (Week 5 schedule page)
  • Complete prelab #4 before your laboratory session next week
  • Write up the discussion, abstract, and introduction sections for your first research paper, due at the beginning of lab next week

Copyright and Intended Use
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Created by David R. Caprette (caprette@rice.edu), Rice University 17 Aug 95
Updated 20 Aug 08