Introduction

Gel electrophoresis is the most widely used method in Biology for separation and analysis of large molecules such as proteins and DNA. The goal of this exercise is to present the basic principles governing all varieties of electrophoretic technique.

Two basic pieces of equipment are required for electrophoresis: a DC Power Supply and a "box" with platinum electrodes to hold the buffer solution and the gel.

The Power Supply transforms the 110 Volt alternating current (AC) supplied by PG&E to an output of direct current (DC) of variable and regulated voltage. The simplest way to describe the power supply is to say that it pushes electrons out through a black wire called the cathode (labeled -) while simultaneously pulling electrons in through a red wire called the anode (labeled +). The output voltage (in VOLTS) indicates how much force is exerted to push and pull electrons. The output current (measured in AMPS) indicates how many electrons flow through the power supply in a given time.

Procedure

PART I:

1. Make sure the power supply is turned off and unplugged.

2. Connect the lead wires from the power supply to the appropriate terminals on an empty electrophoresis box (red to red and black to black).

3. Turn the VOLTAGE to 50. This provides a very strong push to the electrons at the cathode.

4. Plug in the power supply. Turn it on.

What is the AMPS reading?

5. Turn the power supply off.

When you have nothing but air in the gel box, the electrical current is not able to pass from one electrode to the next. Air is not a good electrical conductor and electrons cannot flow from the cathode to the anode. At high voltage (higher than our power supplies can go) the resistance of the atmosphere would be overcome and an electrical spark would jump from the cathode to the anode.

PART II:

1. Turn off the power and unplug the gel box.

2. Fill each side in the electrophoresis box with 50 ml water (either distilled or tap). (The water should not flow over the table inside the box.)

3. Reconnect the box to the power supply and turn the power on.

What is the AMPS reading?

Look carefully at the platinum wire. Do you see bubbles forming on the wire?

4. Turn off the power supply.

Just as before, the current flow could not be measured even at the maximum voltage output because the connection between the electrodes is not made. Therefore, electricity cannot flow from the cathode to the anode. Remember, electrophoresis involves movement of positively and negatively charged ions through the buffer in the box and the movement of electrons through the wires and components of the power supply.

Part III:

1. Turn off the power and unplug the gel box.

2. Fill the gel box with approximately 300 ml of water (either distilled or tap). This time be sure that the water flows from one side of the box to the other.

3. Reconnect the gel box to the power supply and turn it on.

What is the AMPS reading?

Do you see bubbles forming on the platinum wires?

Analysis

1. Will electricity travel through air?

2. Will electricity travel through distilled water?

3. What are the good conductors in parts I, II, and III?

4. What are the poor conductors in parts I, II, and III?

PART IV:

1. Be sure the power supply is off and the box is disconnected from the power supply.

2. Fill the box with distilled water that contains NaCl (0.1 g/liter). (Be sure the salt water solution covers the table in the middle of the box.)

3. Connect the electrodes of the gel box to the power supply and plug in the power supply.

4. Turn on the power supply.

What is the VOLTAGE reading?

What is the AMPS reading?

5. Turn off the power supply.

6. Add a few drops of the red cabbage solution.

7. Turn on the power supply. Set the voltage at 100 V.

Analysis:

1. Carefully watch what happens in the box. Describe what happens.

2. Explain what is happening inside the box in terms of positive and negative charges.

3. Why is it important that the salt water solution connect both sides of the box in part IV?

Conclusion

Write three conclusions about electrical conductors.

How does electrophoresis work?

Extension

Reverse the electrodes. In other words, plug the red wire into the black hole on the power supply and plug the black wire into the red hole on the power supply. You have now reversed the charges. The red wire is now negatively charged and the black wire is positively charged. Add more red cabbage juice and turn on the power.

What do you observe?

How is this different from the results in Part III?

Explain how this difference can occur?