Introduction to electrophoresis

This page is part of the SDS-PAGE lab, which includes these pages:

The whole experiment will be spread over three lab days. Electrophoresis will also be used in several other labs this quarter.

In molecular biology, electrophoresis is a technique for separating macromolecules such as DNA or protein. In Bio 6B, you'll use electrophoresis to find out the results of many of your experiments.

Separation techniques

There are a lot of different molecules in a cell. If you want to study the function of a particular molecule – a protein, for example – you'll need to separate that molecule from all the others. For that reason, molecular biologists and biochemists must master a range of separation techniques to isolate the particular molecules that interest them. Electrophoresis is one of the most important separation techniques for DNA and protein, and you'll use it several times this quarter. You'll do protein electrophoresis first, so I'll discuss it first on this site. 

There are also various other separation techniques commonly used in molecular biology, including chromatography, which we'll use in a later lab.

How electrophoresis works

In protein electrophoresis, you force proteins to migrate through a gel by applying an electric field that pulls on the charged proteins. Positively charged molecules will move toward the negative pole, and negatively charged molecules will move toward the positively charged pole. Uncharged molecules won't move at all. The gel can be any material that is somewhat solid, but has pore spaces large enough for the proteins to fit through. If the pores are a tight fit for the proteins, the larger proteins will move slowly, while the smaller proteins will be free to move faster. 

A protein's rate of movement through the gel will be controlled by:

  • Molecular mass. Larger, more massive molecules move more slowly, because they don't fit through the pores easily.
  • Charge. Strength and polarity of charge influence how fast and which direction a molecule moves. Some proteins are positively charged and some are negative; in most cases, the charge will depend on pH and other aspects of the solution. Using SDS-PAGE, as described below, you'll be able to control the charge on your proteins.
  • Shape. A protein that is tightly folded will seem smaller, and move through the gel faster, than a protein that is loosely folded.
  • Pore size. Bigger pores=faster movement. Smaller pores may allow more precise separations. Pore size varies from gel to gel, but within a single gel, each lane has the same pore size.
  • Voltage. The more voltage you apply across the gel, the faster things will move. Within a single gel, voltage is constant.

Key point: Using appropriate techniques, we can separate our DNA or protein molecules purely on the basis of size (in other words, molecular mass) by holding all the other variables constant. This is true for both DNA and protein electrophoresis.

In Bio 6B, you'll use two different electrophoresis techniques: SDS-PAGE for proteins and agarose gels for DNA.


In SDS-PAGE, proteins are separated purely on the basis of molecular mass. All the other variables are controlled. To understand how this works, you should understand what SDS-PAGE stands for.

SDS stands for Sodium Dodecyl Sulfate – but don't worry about remembering that; just call it SDS. SDS is a detergent, and it is included in the buffers used in SDS-PAGE. This accomplishes several critical things for electrophoresis:

  • SDS helps proteins dissolve so you can run them on the gel (not all proteins are soluble in plain water).
  • SDS helps to denature, or unfold, proteins. This means that the relatively weak hydrogen bonds, hydrophobic interactions, and ionic bonds that maintain the proteins' tertiary shape will be undone, but the polypeptides' primary structure will not be altered. (Don't worry if you don't yet understand primary and tertiary structure; these concepts will be covered in the first lecture unit.) The end result of using SDS is that proteins' shapes will not influence their rate of migration in the gel.
  • SDS sticks to proteins and makes them negatively charged. Since SDS sticks all over the protein, each protein ends up with the same density of charge.
  • In order to make this work, you'll have to pre-treat your protein samples with SDS and include it in your gel. [For today's lab, you may be using Lithium Dodecyl Sulfate instead of SDS; it's still called SDS-PAGE.]

PAGE stands for Polyacrylamide Gel Electrophoresis. You should probably remember this. Polyacrylamide gels are made by starting with a solution of acrylamide and adding a polymerizing agent, then pouring the solution between two plastic plates. The acrylamide polymerizes and forms cross-links, creating a net of large polymers with water-filled pores that are about the size of a protein molecule. When you put your proteins in the gel and pull them through with an electric field, they will be squeezed through the pores.

Polyacrylamide is used because it creates strong gels with a predictable pore size, allowing us to use thin gels and get well-defined separation of our proteins.

In SDS-PAGE, all the proteins have the same charge density and the same unfolded shape. In a single gel, the pore size and the voltage are constant. Therefore, in SDS-PAGE, a protein's rate of migration through the gel is determined solely by size (molecular mass). SDS-PAGE is the most commonly used method of protein electrophoresis. It's the one you'll use in the 6B lab.

Preparing samples for SDS-PAGE

A protein sample for SDS-PAGE begins with a lysate. See the Protein Sample Preparation page for instructions on how to make a lysate from a bacterial culture. Once you have a cleared lysate, you will mix it with SDS sample buffer (or LDS sample buffer, which is the same thing except with lithium instead of sodium ions).  The sample buffer includes several important ingredients:

  • SDS to denature proteins and make them negatively charged.
  • Glycerol to make the sample dense enough to sink to the bottom of the well.
  • Blue dye such as bromophenol, so you can see your sample as you load it. The blue dye will also migrate through your gel a little faster than your smallest proteins. If you run the gel until the dye reaches the bottom of the gel, you'll know that your proteins have spread from bottom to top. The dye in the sample buffer does not stain the protiens, though; you will still need to stain your gel after running so you can see your protein bands.

In addition to the sample buffer, you need to add one more ingredient:

  • Reducing agent to break the disulfide (S-S) bonds that form between the sulfhydryl side chains of cysteine amino acids, forming part of the tertiary structures of proteins. The reducing agent (for example, dithiothreitol) breaks the disulfide bond by reducing  the sulfur atoms. The disulfide bond could potentially re-form by an oxidation during electrophoresis, so we usually add an antioxidant to the running buffer.

After you combine your protein samples with sample buffer and reducing agent, it's important to mix thoroughly and heat the sample so these ingredients can do their jobs.

Agarose gels for DNA electrophoresis

In Bio 6B, you'll use agarose gel electrophoresis to separate DNA molecules by size; this will be the end point of all the DNA experiments you do. Your DNA gel will tell you whether you've got the DNA you're looking for.

The techniques you use for DNA electrophoresis and for protein electrophoresis are different because DNA is different from protein in several important respects:

  • DNA is always negatively charged in solution. With proteins, you had to use SDS to ensure that all proteins had the same negative charge density, but you don't need to use SDS with your DNA.
  • DNA doesn't usually have tertiary structure. With proteins, you used SDS, a sample reducing agent, and heat to unfold the proteins' tertiary structure; you won't need to do that with DNA. DNA usually has secondary structure (the double helix), but not tertiary structure. Circular DNA molecules, such as plasmids, are an exception to this; they will be covered in a later lab.
  • In our DNA experiments, there will usually be small number of bands in each lane -- unlike the protein gels, which will have more bands. Therefore, you won't need the resolving power of the thinner, stronger polyacryamide gels used for protein.

Because of these differences in the molecules and the experiments, your procedures will be different. Sample preparation for DNA gels is easier; you don't need to heat your sample or use SDS or a reducing agent. 

Gel preparation is a little more involved, simply because you'll make your own DNA gels, instead of using premade gels as we did with SDS-PAGE.

See DNA Electrophoresis for the methods.

Agarose vs. polyacrylamide: more detail

Agarose gels are usually have much larger pores than polyacrylamide gels, which makes them suitable for bigger molecules. In the Bio 6B lab, our the DNA molecules in our agarose gels are generally going to be much bigger than the proteins we're looking at in SDS-PAGE. For example, one green fluorescent protein (GFP), which we'll look for in the pGLO lab, has a molecular mass around 27 kiloDaltons. In contrast the pGLO plasmid DNA has a molecular mass around 3300 kD (pGLO is 5371 base pairs long, and one b.p. of DNA has a molecular mass of 618 Daltons). Even a small DNA 900-base pair PCR product, would be 581 kD. That's the main reason why we'll use agarose gels for DNA.

Agarose gels are also inexpensive and easy to prepare, compared to polyacrylamide gels. On the other hand, polyacrylamide makes gels that are much stronger, so it's possible to make very thin gels for high-resolution separations.

You could use a polyacrylamide gel for DNA, and people sometimes do, especially if they need to separate very small DNA molecules that are nearly the same size. You could also use an agarose gel instead of polyacrylamide for proteins. That would work, and it was once the standard procedure, but agarose gels give less-precise results.


Terms & concepts

  • Electrophoresis
  • PAGE (Polyacrylamide Gel Electrophoresis)
  • SDS (Sodium Dodecyl Sulfate)
  • Separation techniques

Review questions

  1. In DNA electrophoresis, DNA molecules are normally separated on the basis of differences in ________.
  2. In SDS-PAGE, protein molecules are normally separated on the basis of differences in ________.
  3. What makes macromolecules move through the gel in electrophoresis?
  4. What determines the speed at which macromolecules move through the gel in electrophoresis? In a single gel, why do some move faster than others?
  5. What controls the charge on DNA molecules in DNA electrophoresis and on protein molecules in SDS-PAGE?
  6. Why do we use different procedures for DNA and protein electrophoresis?
  7. Why do we usually use agarose for DNA and polyacrylamide for proteins?
  8. Compare and contrast the sample buffers for DNA and protein electrophoresis.
  9. Compare and contrast the running buffers for DNA and protein electrophoresis.

Also see the review questions on DNA electrophoresis method and SDS-PAGE.

References & further reading


How SDS-PAGE Works on Bitesize Bio.

A Guide to Polyacrylamide Gel Electrophoresis and Detection from Bio-Rad. This pdf is both an explanation of the principles involved and a catalog of related products sold by Bio-Rad.

More detail

Agarose Gels Do Not Polymerise! on Bitesize Bio.

For more a more detailed explanation of agarose vs. polyacrylamide (far more than you need to know for this lab!), try this article: Electrophoresis of DNA in agarose gels, polyacrylamide gels and in free solution Nancy C. Stellwagen Electrophoresis. 2009 June; 30(Suppl 1): S188–S195.

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