Gel Electrophoresis Explained
Gel electrophoresis is simply identification. Due to its versatility, it has become one of the most important tools of crime scene investigation and protein analysis. Forensic investigators use this tool of genotyping DNA samples to distinguish one human being from another. Gel electrophoresis is commonly used around the world for not only for the investigation of crime scenes but also for identifying missing persons, attaching names to bodies in mass disasters, naming the victims in human rights violations, and in paternity testing.
Gel electrophoresis is an important tool in other laboratory investigations of DNA, and also proteins.
What is Gel Electrophoresis?
Gel electrophoresis is a process where a sample, typically proteins or DNA, in solution is pulled apart or separated inside a mold using electrical charges.
Suppose you want to do a polymerase chain reaction (PCR) to study a single gene. When you do PCR, you make many copies of DNA so you can compensate for the limitations of sequencing methods when you do genomic analysis. Most genomic sequencing methods miss a few segments or have problems with segments of DNA with certain traits. Making copies of DNA makes it easier to error-correct anomalous results.
Or suppose you paste a gene you want to study into a circular piece of DNA called a plasmid for cloning. Maybe this specific gene has therapeutic or academic significance. After using enzymes to cut and paste the DNA into the desired plasmid, you want to make sure that your recombinant DNA has the desired gene in it. The full plasmid will be of a very specific size and recognizable once electrophoresed.
Both of these processes and many other protein analyses use gel electrophoresis to separate target molecules for further analysis. The process sounds really complicated, but the process isn’t hard to understand step by step.
Key points for this lesson:
Gel electrophoresis is a technique for separating DNA fragments by size or proteins by an electrical charge. It doesn’t do genomic sequencing – It prepares the sample for genomic sequencing.
Samples are loaded into indentations called wells at the center or negatively charged end of a sheet of gel, and then an electrical current is applied to pull the samples through the material.
All DNA fragments have the same negative charge per unit of mass, so smaller fragments will move more quickly through the gel and larger fragments will move more slowly through it.
Proteins carry different electrical charges and will travel in whichever direction is dictated by the current in the chamber. More strongly charged proteins will migrate farther than less strongly charged ones.
The distance Proteins travel is a linear function of the electrical charge of the protein.
The distance DNA travels is not a linear function, but a logarithmic function of molecular size. A molecule that is 1/10 the size of another molecule will travel twice as far, a molecule that is 1/1000 the size of another molecule will travel three times as far, and so on.
DNA and some proteins such as Albumins are transparent, so a dye needs to be added to the sample so it can be seen later on in the gel.
How Gel Electrophoresis is Performed
We’ll review some of the basic concepts of gel electrophoresis as we go along to make the reasons for each step clearer.
This technique is a laboratory method for separating and analyzing large molecules, such as proteins, DNA, and RNA. Clinical chemists use it to separate proteins by charge and size, and molecular biologists use it to separate samples of fragments of DNA and RNA by length.
Both DNA and Protein electrophoresis can be performed with the same equipment and in the same manner. Horizontal gel electrophoresis uses an agarose gel, which under a microscope has a mesh-like structure. While vertical electrophoresis uses a polyacrylamide gel. Polyacrylamide gels are a thinner consistency, which can make the viewing the electrophoresis process easier. This technique is based on the movement of charged molecules when they are exposed to an electrical field. This movement occurs across the sheet of gel, or to put it another way, in a gel-medium. But what does electricity have to do with DNA or proteins?
DNA is negatively charged. This is due to the presence of phosphate groups. The DNA helix does not consist just of the four bases adenosine (A), guanine (G), cytosine (C), and thymine (T). Its backbone contains billions of phosphate groups, each with a tiny negative electrical charge. The presence of phosphate groups gives DNA its helical shape.
There are also phosphate groups in the RNA helix, and on other kinds of proteins. Proteins in general have negative charges. Smaller proteins have smaller charges, and larger proteins have larger electrical charges.
Preparing agarose gels is very simple.
Agarose gels are prepared in a horizontal casting tray. The tray will need either tape or removable rubber bumpers covering the two open ends to form the mold for the sheet. Casting trays will have indents for an electrophoresis comb. The combs come in all sizes and colors, they can have teeth on one side or be double sided. These teeth are what will create the wells for the samples to be inserted.
Agarose, which comes from seaweed, will start in a powder form. The powder is dissolved with a pH-specific electrophoresis buffer. Which buffer to use is determined by whether DNA or Protein samples will be used. Simply boiling together agarose and electrophoresis buffer will dissolve the agarose. Then carefully pour the agarose solution into the casting tray like pouring a Jell-O mold. While the agarose is still steaming and liquid, slide the comb into the agarose where the casting tray is indented. The agarose will then need to cool and solidify. Once the gel is ready it will be solid with a firm Jell-O-like consistency. Very carefully remove the comb to reveal the wells then remove the tape or bumpers so both ends of the solid agarose material are exposed. A traditional electrophoresis chamber will have a negative and positive electrode with space between for the material to sit. The electrodes will connect to a removal top and will plug into a power supply.
Set the casting tray, with the agarose still in place, into the electrophoresis chamber. If the wells are closer to one end of the gel, place the wells closer to the negative electrode. Using the same pH-specific electrophoresis buffer, pour the buffer to the chamber until it is just covering the top of the gel.
Carefully insert one prepared sample into each well using a micro pipettor. Many times, the samples will be clear, therefor a stained marker or dye will be loaded into a dedicated well. Once all the samples are loaded, replace the chamber lid, and turn on the power supply to begin the electrophoretic run.
Power supplies can be set to different voltages. It is key to keep an eye on the electrophoretic run. Too high of a current (or voltage) can melt the gel.
Once the stained marker is about one centimeter away from the edge of the gel, turn off the power supply then remove the lid of the chamber.
When you look at the material through a microscope, you see a mesh-like structure. The size of the pores in material is nearly constant throughout. When different-sized fragments are exposed to an electrical charge across the material, the smaller fragments escape the pores faster and the larger fragments take longer to move through the pores.
At this point, it may be hard to see the samples, as they were clear to begin with. There are a variety of stains available to make the samples visible, which does depend on if DNA or proteins samples were used. The stain will bind to the sample to make them visible on a light box or under ultraviolet light. For DNA samples, either Methylene blue or Ethidium Bromide are good choices. While Proteins typically use Coomassie Blue or a color development buffer. As DNA and proteins use the same process for electrophoresis, they too use the same process for staining.
To stain the gel, simply submerge it in the chosen stain for several minutes. Then rinse by submerging in water. Gels stained with Methylene Blue, Coomassie Blue or color development buffer can be placed directly on a light box and can be seen with the naked eye. The samples will be a blueish-purple color. Gels stained with Ethidium Bromide require an ultraviolet light and amber UV-blocking glasses to view.
How to determine the size of the fragments or samples on the gel? Simply compare with the known band sizes or dye sizes loaded into the dedicated marker well. For DNA, a DNA ladder with bands of specific sizes will be used. For proteins, several proteins, or dyes of known molecular weight or charge will be used to create the ladder effect.
Elution can be used to then take the fragment of the sample from the material to be used for further future use. The elution process is very simply just cutting out the fragment from the gel and forcing it through a strainer in a centrifuge to remove the agarose but keep everything inside the pores. After this process, you’re left with a solution of DNA or Protein dissolved in electrophoresis buffer. Elution is done in such a way that the individual fragments of DNA can be used for further downstream processing, such as genome sequencing.
Get Gel Electrophoresis Kits and Experiments
Does all of this sound impossibly complicated? It’s not! Working step by step, you can do electrophoresis. Modern Biology sells the protein, and DNA experiments, as well as electrophoresis packs your students need to begin their learning experience with gel electrophoresis.