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LPS Analysis (lipopolysaccharide analysis)

    Truly wild strains of the intestinal bacterium E. coli possess many characteristics not found in the common K-12 laboratory strains. There is a division of thought between Europe and the USA: European bacteriologists say that the identifying characteristics of bacteria are not affected by growth conditions, while Americans disagree. Using some biochemical techniques, we shall be inspecting the influence of environmental conditions on one of the immunologically important surface layers of several E. coli isolates from the wild.
    Mnemonically, we can undress E. coli. Its exterior outer layer is acidic polysaccharide called capsule (or K-antigen for Kapsul in German). The next "underwear" layer is lipopolysaccharide (or LPS or endotoxin or the O-antigen for Ohne Hauch). This is the layer we will be studying in depth. Another exterior feature on E.coli are its flagella (or H-antigens). Beneath all of these are its "skin", its cell membranes and cell wall. As with humans, wearing flagellar tassels, outwear and underwear are nice but not necessary. Common K-12 lab strains are "streakers," and thus not appropriate for our current study. Thus the wild strains are employed.
    Quantitative biochemistry generally has two strategies. The first is to isolate the compound from all the other cell matter and then measure it. The other strategy is to find a reagent that specifically reacts quantitatively with the biochemical of interest. We shall be following the second strategy. Unlike acidic capsular polysaccharide, LPS's polysaccharide contains many vicinyl hydroxyls, and you have seen that these are target for periodic acid oxidation, cleaving them to form aldehydes that then can reduce silver ions to silver in a fashion not unlike in photographic development.
    We shall thus be taking about a colony's worth of cells and lysing them in detergent, and then electrophorese the zillions of cellular components in an aqueous plastic gel (polyacrylamide). Of the zillions of resulting separated componest, periodate will attack only those having LPS and making aldehydes that then will reduce Ag+ to Ago, with will form visible bands in the gel. From electophoresing lysates from cells grown under various conditons we shall see whether the European view is correct. What is more, since LPS is a major vaccine component, we might gain some insights into improving vaccines, and perhaps also understanding the value of fever.
    You will see that much of the technique is similar to DNA sequencing, and other types of protein electrophoresis. The reason for presenting this rather that those is that LPS/SDS-PAGE is an end in itself and can demonstrate the power of the fundamental procedure without your getting bogged down in many other subprocedures, and yet will reveal marvels that can be applied to save lives by the millions! (Which it is!)

    The figure next to the title illustrates a well-attired E.coli. Then click for a closer look at the generalized structure of LPS.

    There are many pages of directions for setting up, running, and developing the SDS-PAGE electrophoretigrams. Print them out!