Most
molecules are electrically
neutral, but some
important biological molecules, including
proteins,
DNA fragments and many
natural dyes, carry a net
negative charge when they are in
solution.
Electrophoresis cleverly uses a weak
electric field to force such charged molecules to
drift through a medium that separates them by offering differing amounts of
resistance to
motion.
You can easily see this
phenomenon in
action when you place a droplet of dye on a strip of
blotting paper that has been wet by a
conductive fluid, such as
salt water. When the ends of the
paper are connected across a
battery, a voltage is set up, which drives the
charged dye molecules through the
paper.
Positively charged particles move toward the
negative terminal, whereas
negative ones move toward the
positive terminal. Usually, larger
molecules have a more difficult time than smaller ones in passing through
paper fibers, so the smaller
molecules drift faster.
Thus, over
time, the different
molecules in a
mixture will tend to sort themselves by size.
It takes
only a few minutes to set up a basic
apparatus. From a large
coffee filter cut a rectangular strip of
paper that is about one
centimeter (about [half an inch) wide and about 15
centimeters (
six inches) long. Place this
paper band inside a flat
glass pan or
cooking dish.
Roll each end of the
paper strip around a
nail, and use an
alligator clip to secure it.
Wire the clips to five
nine-volt batteries connected in
series.
To make the
conductive solution, mix about
100 milliliters (
four ounces) of
distilled or
bottled water with 1.5
grams (about a
quarter teaspoon) of
table salt. Then thoroughly wet the
paper, including the
nails, with the
salt solution, but don't add so much that the paper is
submerged in a puddle.
To begin, use a
toothpick to place
droplets from several different
hues of
food coloring in a
line, then
connect the
electrodes. The
colors will rapidly spread into streaks as the
pigment molecules migrate toward the
positive electrode. Next, mix two of the dyes, say,
red and green, and run a
tiny splotch of the combination. After about 20 minutes, the colors should begin to separate. The same technique can be used to separate other
molecular mixtures.
So here's how to find out if two plant species use the same molecules as pigments. First, crush the flowers and immerse them in clear isopropyl alcohol, letting the solids settle. Pour off each of the resulting color-tinged liquids into separate containers and then concentrate them by letting the alcohol evaporate. Once the alcohol is nearly gone, dissolve the pigments in a few drops of the salt solution you made earlier.
Next, line up three tiny dots of pigment on a strip of soaked filter paper by placing a p
ure sample from each plant on the outside and an
equal mixture from both in the center. Then connect the
batteries. If the outside dots separate into different sets of colored swaths and the center streak appears to be a combination of the outer ones, then you know that different pigments are involved. But if all three dots form the same pattern, then both plants probably rely on the same molecules for color.
Note that the
salt ions will also
drift toward the
electrodes, where they will quickly create a layer of
tarnish that
impedes the flow of electricity. So after each run, you will have to
scrub the electrodes. As all this cleaning rapidly becomes tiresome, you might try to replace the steel nails with another conductive material that does not tarnish as quickly--stainless-steel wire or
aluminum foil, for example. Small pieces
gold or
platinum wire or
chain work especially well.
Although many great discoveries have been made using paper-based
electrophoresis, this simple method does have a big drawback: the molecules tend to get caught up in the fibers of the paper. This complication explains why even pure dyes form streaks instead of remaining well-defined dots as they move along. So these days biologists often replace the paper with a more uniform material called
agarose--a clear substance with the consistency of stiff gelatin. The DNA "fingerprint" patterns you may have seen are produced by electrophoresis on such a gel. Each of the individual lines in the fingerprint indicates strands of DNA of a certain length. Compared with results with paper, the degree of separation possible with a gel electrophoresis is amazing.
You can quickly fashion a gel-based
electrophoresis unit from any small, rectangular container that is waterproof. I used the bottom of a plastic soap dish. Bend some
aluminum foil over the two shorter sides to serve as electrodes and then pour enough of the
hot, liquid agarose into the dish to cover it with a half-centimeter layer. Because your gel must contain reservoirs to hold the concoctions you wish to separate, cut out a comb shape from a Styrofoam tray--the kind used to pack meat at the grocery store--and suspend it so that the tines penetrate the liquid agarose but don't poke through the bottom. Let the gel set before carefully removing the comb. This maneuver should produce a series of nicely spaced wells for your samples. Now add enough of the salt solution to cover the gel and keep it from drying.
With an eyedropper, place your test substances into the wells, rinsing the dropper thoroughly between samples. To start your experiment, just connect the
aluminum foil to
your batteries with
alligator clips, with the p
ositive terminal attached to the side opposite the wells so that the negatively charged molecules have some room to move. Don't worry if you notice some bubbling along the foil as water molecules are split apart by electrolysis. And don't be concerned if the
color of the pigments changes (a common effect of
altered pH). Because of its tendency to tarnish, you will have to replace the aluminum foil when you renew the agarose after each run.
Most of this is from
scientific american. It really works.
Narf.
Toodles, adamwolf