(An Overall View of a Possible Experiment)

    One of the many powerful tools for the study of biochemicals is electrophoresis. Since we might simplisitcally conceptualize our bacteria as being sacks of biochemicals, we should hope that this tool would be valuable to our insights into the microbial world. Using SDS-PAGE (sodium dodecylsulfate-polyacrylamide gel electrophoresis), we shall inspect the lipopolysaccharide, LPS, fabric of the "sack" itself. Like any fabric, it is made up of lint segments ranging from small to large, and each bit of lint has a specific size. Using SDS-PAGE, we can inspect the size distributions of the population of lint molecules. A typical LPS-ladder is shown to the right. Later, once the method is mastered, it will be invaluable to you in providing new information into environmental effects on these size distributions. But this week is acquaintance week, and you will learn to master the mechanics of setting up the system, and preparing the desired cell-types. Next week, we shall be doing the electrophoresis, and then staining the gels so as to visualize the size distributions and observe the effects that environment had on the cells' LPS.

    DAY #1: PREPARATION OF DESIRED CELL-TYPES. Each group has a number of bacteria in its collection. Make TWO L-agar plates, and inoculate the plates in spots with each of the several bacteria that you want to test with SDS-PAGE. Leave the plates to grow overnight- one at room temperature and the other about about 42°c.

    DAY #2 (must not be later than the next day): HARVESTING OF CELLS AND LYSIS.
Why not after the next day? It has been found that the more vigorous the cells, the higher the molecular weight are its LPS molecules.

1. Label some 1.5 ml Eppindorf tubes, and add 400 μL of highly purified water to each tube.
2. Using a sterile loop, pick up a small blob of each bacterial colony from plates inoculated previously.
3. Suspend and homogenize these blobs in the water in the little tubes.
4. Add 400 μL of "2X Lysing Buffer" (not "1X Lysing Buffer") to each tube
5. Close the caps of the tubes and give them a good shake.
6. With the little tubes half immersed in water, boil for 15 minutes.
7. They can then be stored for months at room temperature.

    ELECTROPHORESIS. The instructor will demonstrate the assembly and loading of the apparatus. Each group will then make up the acrylamide components that will shortly set as an aqueous plastic called polyacrylamide. Several of these reagents are mildly carcinogenic (but less so than secondary cigaret smoke). Wash hands after preparing a gel. Once the gel has set, you will then load your blue samples, and electrophorese for about 4 hours. (This means someone in your group must come back to lab later today.) The gel will be extracted from the apparatus and immersed in a fixative overnight.)

    DAY #3 (must be next day): VISUALIZATION. The gels will then be subjected to a protocol called Silver Staining, which is related to photographic development but uses a chemical activator rather than light. At the end, the developed gels will be soaked at least overnight in a dehydrating solution prior to preservation.

    HINTS about what you see: discrete bands are of single sized molecules; smaller molecules move further; more highly charged molecules move further. Movement is thus a charge per mass phenomenon.

    DAY #4: PRESERVATION. In frames, the gels will be sandwiched between cellophane and allowed to dry. The framing takes about 5 minutes. After 36 hours of drying at room temperature, the cellophane may be cut from the frames, written upon with marker, taped into notebooks, etc., xeroxed, photographed. If you have beginner's luck on your side, you might get a publishable result on the first try.