Biotech basics: dye electrophoresis deep dive

We often think about agarose gel electrophoresis as a technique that is used to separate DNA fragments, but the truth is that this technique is incredibly versatile and can be used to separate all kinds of molecules. Gel electrophoresis can be defined as a technique that uses electricity and a porous gel matrix to separate molecules into discrete zones, or bands, based on its physical properties. This includes charge of the molecule, its size, and its shape. We’re not limited to separating DNA using electrophoresis – the first electrophoresis experiments focused on ions and proteins. (If you are more interested in learning more about the history of electrophoresis, check out our past blog post).

For DNA, we often think that the size is the most important thing for separation by electrophoresis – after all, that is what we are using the technique to analyze – but the negative charge of DNA is what allows the electrical current to push the nucleic acid through the gel. And this becomes important when separating dyes using electrophoresis. So, what you’ll see here in this picture are three commonly-used biological dyes are shown here – Crystal Violet, Orange G, and Xylene Cyanol.

  • If you’ve done a Gram stain on bacteria, you’ve used Crystal Violet. This stain is responsible for the purple color of Gram-positive bacteria.  The stain sticks to the bacterial cell wall. 
  • Orange G is used to stain keratin in the Pap stain, which identifies cancerous cells in cell smear preparations.   
  • Xylene cyanol is commonly used in agarose gel loading buffers, to monitor the progress of electrophoresis.

At first glance, they’re similar molecules.  They’re all around the same size, and they all have aromatic rings.  When we look more carefully, their chemistry is actually different – we have some different bonds, some different functional groups, all which affect the charge of the molecules.  So, crystal violet has an overall positive charge, and orange g and xylene cyanol have an overall negative charge.  By virtue of its side groups and bonds, Orange G has a larger negative charge than xylene cyanol. 

So, what does this mean when we add the samples to the wells and apply current?  The dye molecules have an overall net charge which influences their movement through the gel.  Because molecules with different charges travel at different speeds, they become separated and form discrete “bands” within the gel.

Some of the dyes are negatively charged (like DNA) and will move through the gel towards the positive electrode.  Dyes with greater net charges will be more strongly drawn to the electrodes.  So, for example, we looked at two charged dyes before, xylene cyanol and orange g.  Because of its larger charge, the orange g dye moves through the gel more quickly.

In contrast, Crystal Violet is a positively charged dye.  Instead of traveling to the positive electrode, it is going to travel to the negative electrode.  If you’re used to seeing DNA gels, this is going to look like the sample is running in reverse! But, it’s doing exactly what it should be doing based on its chemical properties.

Now, to connect back to standards, a deeper discussion of the technique of gel electrophoresis addresses many disciplinary core ideas and topics as described by the High School Life Science Standards. If you’re teaching biology, many of the standards focusing on life sciences are applicable — structure and function of DNA and heredity, and variance of traits. But the dyes do allow us to bring in additional learning standards

  • NGSS DSI PS2, through an exploration of the ways that objects can interact with one another; specifically, the interactions between dyes, the agarose matrix, and the electric field used in electrophoresis to separate molecules by size and charge, bring physics into our biotechnology classroom.
  • Cause and Effect: how different dyes separate in a gel, and how the migration of dyes is influenced by different electrical currents. 
  • SEP1: Asking Questions and Defining Problems, through the evaluation of the experimental data and the critical reading provided background information.
  • SEP6: Constructing Explanations and Designing Solutions, through the understanding of the properties of dyes and the principles of electrophoresis.

To learn more about dye electrophoresis, check out our past live stream where we discuss it in detail (slides available for download too!)

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