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Laboratory
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Recordkeeping, Writing,
& Data Analysis

Laboratory
Methods

Overview
Microscope studies

Flagella experiment
Laboratory math
Blood fractionation
Gel electrophoresis
Protein gel analysis
Mitochondria
Concepts/ theory
Overview
Keeping a lab notebook
Writing research papers
Dimensions & units
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Examples of graphs
Experimental error
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Overview
Principles of microscopy

Solutions & dilutions
Protein assays
Spectrophotometry
Fractionation & centrifugation

differential centrifugation

Tissue Fractionation

Just as a whole number can be considered to be the sum of individual fractions, tissue can be treated as the sum of individual parts that we also call fractions. We can take tissues apart, that is, fractionate them, in order to obtain fairly pure samples of the components that we need for study. Tissue can be fractionated in a number of different ways, depending on what part we wish to isolate. By comparison, the number 1 can be expressed as 1/5 + 2/5 + 2/5. We can divide it differently, for example, 1 = 1/2 +1/16 + 7/16.

The purpose of tissue or cell fractionation is to obtain a pure sample of part of the original whole, such as mitochondria, plasma membranes, DNA, RNA, soluble proteins, or even a specific macromolecule. For example, whole liver includes connective tissue, arteries and veins, fatty deposits, hepatocytes, etc. We can choose to isolate the hepatocytes (liver cells) from the rest of the material, obtaining two fractions such that

whole liver = [hepatocytes] + [non-hepatocytes]

We can further fractionate the hepatocytes. For example,

hepatocyte suspension = [liver mitochondria] + [cell nuclei] + [vesiculated membranes, remaining organelles, and cytoplasm]

Another example is the fractionation of whole blood, which is considered to be a tissue.

whole blood = [plasma] + [red cells, white cells, and platelets]

Methods for the fractionation of tissue often start with homogenization using a blendor, grinder, or some other mechanical device, followed by differential centrifugation. Suspensions of cells that are obtained from whole tissue must be lysed (burst open) in order to release their contents, unless lysis is accomplished by the homogenization itself.

We conduct a simple fractionation in the teaching lab in order to obtain fairly pure preparations of red cell (erythrocyte) plasma membranes, with their associated proteins. Homogenization is not necessary, since whole blood treated with anticoagulant remains a liquid. We first use centrifugation to separate blood plasma from the formed elements (red and white cells, and platelets). We then lyse the red cells and separate the membranes from the cytoplasm.

Another scientist may read your research paper, and may want to know how much material he/she can expect to obtain by using your methods. The investigator may want to isolate another part of the tissue, not necessarily the part that you studied. Therefore, when we report the results of a tissue or cell fractionation, we need to describe how much material we obtain for each fraction, for a given amount of starting tissue. We call that the yield, and describe the yield as total amount of protein in that fraction.

Total protein in a volume of whole blood = [total protein in plasma] +[ total protein in white cells and platelets] + [total protein in red cell cytoplasm] + [total protein in and attached to red cell membranes]

To obtain total protein in a fraction, we need to know two things, namely the total volume of the fraction and the protein concentration for the fraction. Nearly all fractions are suspensions of particles or are solutions, thus we need only record a liquid volume in order to obtain the first bit of information. In order to obtain the protein concentration we need to save a sample (called an aliquot and pronounced al'-i-kwat) and conduct a protein assay on that sample. It is seldom necessary to save the entire fraction - we only need a sample.

yield = [total volume of a fraction] x [protein concentration in the fraction]

Yields are typically reported in a table, with columns for (1) name of the fraction, (2) volume of the fraction, (3) concentration of protein in the fraction, and (4) yield. We report only yields of the principal fractions, that is, those that contribute most of the protein. Thus, in the blood fractionation we don't worry about the small contribution from white cells and platelets, which are lost anyway during the various wash steps.

The yields that we report are invariably just rough estimates. Any fractionation process results in loss of material, so that often the total of all yields does not add up to the amount of starting material. What is even more frustrating for inexperienced investigators is that sometimes the total of all yields adds up to more than the starting amount! That is because all protein assays differ in their sensitivity to concentrations of different proteins, so that the sensitivity of an assay to protein concentration in two different fractions of differing composition can differ by two-fold or more. If that happens, simply report the yields as calculated and explain the inconsistency in your discussion. Do be wary, however, of extreme inconsistencies. If you wind up with, say, ten times more protein than you started with, you might want to check your work.

One more thing - because yields are inherently inaccurate, we don't try to obtain an extremely precise estimate of total protein in any given fraction. Generally, if you use centrifugation to separate the liquid portion of a suspension (the supernatant) from the semi-solid pellet, you only need to take a sample of the supernatant and a sample of the pellet after you resuspend it. If you continue to wash the pellet free of contaminants, don't worry about the "extra" protein that you are throwing away.