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Guide to the study

• membrane organization
  
• overview of the study
• research paper

Lab part 1

• components of blood
• blood and red cell fractionation

Lab part 2

• introduction to SDS-PAGE
• preparing gels
• preparing samples
• assembly, loading, running gels

Lab part 3

• gel images
• gel analysis
  
• molecular mass standard curve
• measuring relative mobility
• "Hall of Shame"

Selected methods

• tissue fractionation
• centrifugation
• Bradford protein assay

Characterizing Proteins Associated with the Mammalian Red Blood Cell Membrane

If you want to know how something works, you have to take it apart. I'm sure that as a kid you did that with a flashlight, machinery, or some electronic device, and you may have even gotten into trouble because you could not put it back together. We need to take organisms, tissues, cells, and organelles apart in order to discover how they function, and for certain we cannot put them back together, although we want to learn to do just that.

Overview of the study

Our hypothetical plan is to develop a strategy to repair genetic defects in the red blood cell (erythrocyte) cytoskeleton of individuals. Erythrocytes and other formed elements of blood are derived from adult stem cells found in the bone marrow. Our strategy will have us culturing and genetically engineering bone marrow stem cells to repair genetic defects, then transplanting the altered cells back into the original donor. Funding comes from the National Institutes of Health (N.I.H.), which has called for proposals to address a new threat to the public health (also hypothetical), namely a fatal form of spherocytosis that has cropped up in clusters in different parts of the country.

Spherocytosis is characterized by defects in the membrane associated proteins of erythrocytes that cause them to circulate as spheres rather than as biconcave disks. To work with these proteins we need a reliable means of isolating them from whole blood and cultured material, and a means of identifying them. A quick way to see what proteins you have in a sample is to separate and visualize individual polypeptides by gel electrophoresis. The method of electrophoresis called SDS-PAGE is not only a quick and dirty way of quickly estimating the protein composition of a sample, but it also provides a basis for more specialized identification/purification of proteins such as Western Blotting, peptide mapping, two-dimensional electrophoresis, or electro-elution of polypeptides.

Cell culture requires that we grow cells under carefully controlled conditions in complex (and expensive) media. To obtain enough material for our initial characterization it is more practical to isolate erythrocytes from whole blood. For that matter, we must test our cultured erythrocytes to determine the extent to which they are structurally and functionally similar to erythrocytes in whole blood. It is common for cultured cells to become altered, limiting their relevance to normal cells in intact tissues. We will start with whole blood drawn from a canine donor. We characterize the membrane associated proteins as far as we can using SDS-PAGE alone. This initial characterization will give you a road map of sorts so that you will be able to locate proteins of interest as is necessary to meet your project goals.

The full rationale behind this research project is described in the accompanying presentation. This work is an example of applied science, not pure science. In fact it is more of an engineering project. To oversimplify a bit, a scientist seeks new knowledge about nature itself, while an engineer seeks to solve specific problems using what we currently know. In science there is just one correct answer. In engineering one might take any of several approaches to a problem, thus there are multiple "correct answers." There is quite a bit of overlap, of course. An engineer might very well conduct experiments to acquire new knowledge when what we currently know is not sufficient, and a scientist may indeed engage in applied research to address specific problems.

The initial work could easily be done in a day by an experienced researcher, although staining usually takes an overnight incubation. For practical purposes a teaching lab should spread the work across three days (three weeks for a course that meets one afternoon/week).

First week – Blood fractionation

• separate red cells from whole blood
• lyse red cells and isolate membranes
• determine protein concentration in each fraction
• freeze an aliquot of each fraction
• [optional] preparation, staining, and examination of a blood smear

Second week – SDS-PAGE

• dilute and denature aliquots for electrophoresis
• complete preparation of SDS gels
• load, run, and stain gels

Third week – Gel analysis

• calibrate gels using internal standards
• identify lanes corresponding to specific aliquots
• identify important polypeptide bands associated with the membrane fraction
• identify bands corresponding to hemoglobin
• estimate relative molecular mass for relevant polypeptide bands
• characterize bands by staining intensity, qualitative characteristics of bands
• atttempt to identify (name) bands of interest
• critique the gels