Biotechnology/Bioinformatics Discovery! Project

 

Demystifying Electrophoresis

Lessons in chemistry, biology, physics

 

Electrophoresis is used in biotechnology as a separation method.  Molecules move by attraction to an electric charge, and typically they move through a porous gel that allows smaller molecules to move faster than larger molecules.  Thus, the typical gel electrophoresis experiment separates molecules by charge and molecular weight (size) and shape.  Applications of electrophoresis include sorting mixtures of proteins or DNA molecules for identification purposes (like in forensics).  This series of experiments is designed to deconstruct the process of gel electrophoresis.

 

For a good animation and illustration of this process, visit http://learn.genetics.utah.edu/units/biotech/gel/

 

I.                   What Goes on During Electrophoresis?

 

Let’s explore what goes on in electrophoresis with just the gel box and power supply, leaving out the gel for now:

 

When the power is turned on, there is a negative pole (black) and positive pole (red).  Bromothymol blue is a pH indicator which is yellow in acid conditions, blue in basic conditions.  Predict what will happen to the color of the bromothymol blue solution at the negative pole and at the positive pole when hydrogen ions from water cluster at one pole and hydroxide ions from water cluster at the other pole::

 

Let’s do some experimenting:

Trial 1

1.  Take an empty electrophoresis chamber (box) and fill the two wells in the chamber with bromothymol blue solution (enough to cover the platinum wire at the bottom of each well of the unit, but not enough to cover the platform of the chamber between wells).  Place the box on a piece of white paper. Note--not all electrophoresis chambers have wells that are lower than the platform for the gel. If your chambers don't have these lowered wells, you can not do trial 1 very effectively -- skip on to trial 2.

2.  Note the color of the dye in the chamber at the start – record in table below.

3.  Close the chamber with the top piece, and connect red and black power cords to power supply (red to red, black to black, same channel). 

4.  Turn on power, set voltage to about 100 Volts. Leave undisturbed for 2-3 minutes.

5.  After a few minutes, note the color changes occurring in the electrophoresis chamber.  Record in table below.

 

Trial 2

  1. Disconnect from power source, open box, and add enough bromothymol solution to completely cover the platform that connects the two wells.  Close box, reconnect to power supply.  Run for 5 minutes.  Record results in table.  Also note the relative amounts of bubbles at positive versus negative end – do you see a difference?

Trial 3

  1. Disconnect from power source, open box, and sprinkle some table salt (just a pinch) into the fluid, swirl to mix, close box, and reconnect power supply.  Record results in table.  Note again relative amounts of bubbles at positive versus negative end – any difference?

 

 

 

Trial 1

Trial 2

Trial 3

Starting dye color

 

 

 

Dye color at neg. electrode (black)

 

 

 

Dye color at pos. electrode (red)

 

 

 

 

 

 

 

Analysis of results:

  1. Why in the first trial was the color change minimal? 

 

 

  1. Why was the color change more apparent in trial 2?

 

 

  1. How do you think the addition of salt accounts for the results in trial 3?

 

 

 

 

4.  Was there a difference in the relative amounts of bubbles at each pole?  Yes   No    If you noticed a difference, which pole had more bubbles?  If water is undergoing electrolysis, it breaks down into hydrogen gas and oxygen gas, hence the bubbles.  How does the formula of water, H2O or HOH, tell you where to expect more bubbles?

 

 

 

5.  When you open the box, can you smell the presence of another gas?   Any guesses as to identity? 

 

II.                Agarose Gel Electrophoresis with Dyes

Agarose gel electrophoresis makes use of the electrophoresis chamber and power supply to separate molecules by charge, with the added feature of a gel.  By forcing the molecules through a gel made of a sieving compound like agarose, you can now  simultaneously separate the molecules by size and shape.  All negatively charged molecules will move towards positive pole, and vice versa, but the smaller molecules will move through the agarose mesh network much faster than larger molecules.  In this experiment you will use gel electrophoresis to separate different dye molecules.  Using dye molecules allows visualization of molecule movement as the process occurs.

Materials and Equipment

For each team:

Various dye samples in microcentrifuge tubes:

  • Micropipettes and tips to load dye samples
  • Electrophoresis units and power supplies
  • 0.8% agarose gels prepared with TBE buffer - with sample wells in center of gel

 

  Loading the samples

  1. Follow data sheet below as to where you will load the specific dye samples (A-F). Place the gel on its casting tray into the gel box and add TBE buffer to the gel chamber till there is about 2-3 mm buffer over the top of the gel.
  2. Load the numbered dye samples into the wells located in the middle of the agarose gel. Use a different tip to load each sample dye.  Load 10-15 ml per well.
  3. Connect electrophoresis unit to power supply (red to red, black to black), being careful not to bump your gel. Plug the power source into an outlet.
  4. Turn on the power supply, and set voltage to ~100-125 V. The dyes will start moving toward either the negative or positive pole according to their charge.
  5. Electrophorese samples for ~10 minutes. Turn off power supply, disconnect power cords from the chamber, and remove top of electrophoresis chamber.

Analyzing the gel

  1. Before you remove gel from chamber, mark the positive end of the gel by cutting off a small corner of the gel. Now carefully remove casting tray with gel. Place gel in large weigh tray for viewing.
  2. Mark the gel diagram below with + for positive end and - for negative end.
  3. Use the gel diagram below to indicate banding pattern results of the samples - use colored pencils if available.

             
    WELL
    		 DYE CHARGE observed
    A Methyl Orange  
    B Bromophenol Blue  
    C Xylene Cyanole  
    D Pyronin  
    E Safranin O  
    F Unknown  


               gelimage                

 

Questions

  1. Which dyes have a positive charge?   Justify your answer:

 

  1. Which dyes have a negative charge?  Justify your answer:


  2. Which dyes do you think are present in the unknown sample? How do you know?

 

  1.  Can you hypothesize which dyes are larger or smaller in molecular weight in comparison to each other? 
  2. Based on results from your first experiment with electrophoresis, what do you think might happen if the agarose gel was prepared with water rather than TBE?

 

Credits:  Some of the ideas for this laboratory exercise came from the University of Arizona Biotech project, CEPRAP at the University of California Davis , and Access Excellence.

 

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