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Sorting Molecules with Electricity

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by Shawn Carlson
December, 1998

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THE MOST WONDERFUL PRIVATE garden I have ever seen is tucked away behind a modest house in La Jolla, Calif., not far from where I live. The gardener is a British-born psychology professor and dear friend who sends me home with fruit and flowers each time I visit. Recently I noticed that two of his plants, though very different in shape, produced flowers of the exact same shade of purple. This observation made me wonder whether the two species might be related.


Figure 1: SOAP DISH with a layer of agarose gel permits complex molecular mixtures to be separated by electrophoresis.

One normally traces evolutionary connections by identifying physical similarities between species. So I decided to extract and isolate the pigments in the two flowers so that I could compare them in detail. That process is actually much easier than it sounds. In fact, using a simple technique called electrophoresis, I could carry out the experiment in about an hour for very little money.

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 pure 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 of gold or platinum wire or chain work especially well.


Figure 2: PAPER STRIP CUT FROM A COFFEE FILTER and soaked with a salt solution provides a medium for electrophoresis.

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.

September's Amateur Scientist column explained how to extract DNA from living tissues. Unfortunately, the extracted material must be subjected to sophisticated laboratory manipulations using expensive reagents before a fingerprint can be created. But similarly diagnostic patterns can be made using plant pigments. Indeed, complex pigments often separate so cleanly that the results are just as stunning. After about 20 minutes, you can often isolate virtually every molecule involved in such a mixture.

Although ordinary gelatin does not work well, I'm told that a food additive called agar-agar may and that it can be found in Chinese food markets. But I suggest that you spend $25 and purchase enough agarose gel for about 40 experiments from Edvotek, an educational biotechnology company in West Bethesda, Md. (301-251-5990).

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.

The secret to successful electrophoresis is in the buffer solution. If it is too conductive it will carry too much current and heat and distort the gel. Walt Allen of the Foundation for Blood Research in Scarborough, Maine submitted the perfect recipe for the buffer: mix one 1.5 grams (one quarter teaspoon) of baking soda to 250 milliliters (ten ounces) of tap water.

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 [see illustration above] 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.

For the separation to take place, the gel must contain ions that can conduct electricity. To add the ions, make the gel with the buffer. Allen's recipe mixes 1.5 grams (one quarter teaspoon) of the agarose gel into 50 milliliters (two ounces) of buffer.

Next, the pigments need to be dissolved into what's called a loading solution before they are placed in the wells. Allen dries the pigments, then mixes them solution consisting of three parts Glycerol and seven parts buffer.

In addition, Allen makes the following recommendations: Don't let the gel occupy the entire container. Rather, pour the gel with Styrofoam fit in the ends of the soap dish. Allen uses Styrofoam from one centimeter thick sheet of packing material to fit in the ends. Once the gel has set these are removed (after removing the comb) so that the gel sits in the middle of the container.

Second, cover the gel with buffer to a depth of 3-4 mm filling the buffer reservoirs at each end of the gel (where the Styrofoam was removed). This keeps the gel from being distorted by the hydrolysis that takes place at the aluminum foil electrodes.

And lastly, use an eye-dropper to drop the sample into the wells. You do this by just penetrating the buffer surface and allowing the glycerol-thickened sample to fall into the wells. When using a pigmented sample this is not difficult to do since you can see it drop and any that does not fall in the well will diffuse away in the buffer. Make your wells small (about 2 mm thick) to keep the sample volume down and concentrated at the bottom of the wells.

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 positive 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.

Electrophoresis is a cornerstone of molecular biology. Armed with this technique you can isolate the basic stuff of biology for further exploration. There are far too many living systems for professionals to study them all, and so there are many discoveries waiting for the ambitious amateur armed with this technique, a textbook and some perseverance. So why not get to work!

 

The Society for Amateur Scientists has joined forces with Edvotek to create a complete gel-based electrophoresis unit for kitchens and classroom labs. Send $55 to SAS, 5600, Post Road, #114-341, East Greenwich, RI 02818, or call the society at 1-401-823-7800. You will find more information about this and other articles from the Amateur Scientist on the World Wide Web.

 

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Wondering where you can purchase the science supplies needed to perform this Amateur Scientist project? At WARD’S you can find everything you need need for studies in the biological and environmental sciences.

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