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While the specific steps of gel electrophoresis may differ somewhat between running DNA/RNA gels and protein gels, overall, the steps are the same. In this post, we’ll go over the five steps of gel electrophoresis.

Table of contents

What is gel electrophoresis?

Gel electrophoresis is a method used in the lab to separate DNA, RNA, or proteins from one another. The molecules of interest are forced through a porous gel by an electrical current, with one end of the gel being positively charged and one end being negatively charged. This results in negatively charged molecules, like DNA and RNA, traveling toward the positive end of the gel. Since proteins can have a variety of charges, they must be neutralized using sodium dodecyl sulfate (SDS) to ensure the molecule separation is not affected by charge but is only due to size. SDS also denatures proteins, preventing variations in molecule shape from affecting migration patterns.

Due to the size of the pores in the gel, larger molecules do not travel as far as smaller molecules, allowing for size separation to occur. In the end, the separated molecules can be visualized as bands1.

What do you need for gel electrophoresis?

First, let’s go over everything you need for gel electrophoresis. While there are different types of gel electrophoresis, the same tools are required for each type2What you’ll need to begin gel electrophoresis:

  • Gel box

    The gel boxes differ depending on the type of gels being run. The optimal choice for DNA and RNA separation using agarose gels is horizontal gel electrophoresis. Vertical gel box systems, like the Azure Aqua Quad Mini Cell (shown below), are the best for separating proteins using polyacrylamide gels. The Aqua Quad Mini-Cell is designed for running 1–4 precast or handcast gels (cassette size 10cm x 8cm). It features locking side fasteners that provide a tight seal to ensure rapid and easy electrophoresis.

Loaded gel in electrophoresis for SDS-PAGE
Protein samples loaded into a gel inside an Azure Aqua Vertical Gel Running System
  • Gel

    Either pre-cast or hand-cast, with wells at the top of the gel for the samples to be loaded into prior to their migration through the gel. The gel is submerged in running buffer containing salt ions which conduct the electrical current throughout.

  • Running buffer

    Research the type of running buffer you will need and prepare it ahead of time. Buffers can often be purchased; however, they are also often made in bulk in the lab.

  • Power supply

    A power supply, like the Azure Aqua Power Supply (shown below), provides the electrical current through cables that connect to the positive and negative terminals of the gel box. It is equipped with four power connections that allow you to run multiple experiments at once, and can control constant power, current, or voltage.

Azure Aqua
The Azure Aqua Power Supply is a universal power supply that is designed for powering electrophoresis and transfer modules.
  • Visualization system

    Because DNA and proteins cannot be identified with the naked eye, there must also be a way to visualize them after separation. After transferring the gel to a membrane, use an Azure Imager, or another digital imaging system for further analysis.

Azure Imaging Systems allow you to stop wasting money on film. These modular, multichannel imagers are capable of UV, color imaging, blue-excited DNA dyes, silver-stain, Coomassie gel, fluorescence imaging, and more. Azure Imagers are come equipped with visible fluorescence, visible light, and UV excitation channels and are fully upgradeable to access a wide breadth of applications.

Compare each model in the Azure Imaging System lineup by clicking here.

Scientist choosing settings on Azure 600
The Azure 600 is the only system that offers two channel laser based IR detection, chemiluminescent detection with the speed and sensitivity of film, and the ability to image visible fluorescent dyes, standard EtBr and protein gels.

For quick, simple confirmation of the presence of the bands, a handheld UV light or light box can also be used. To detect individual proteins, antibodies specific to the proteins of interest must be used. Antibodies can be designed to be detected by either chemiluminescence or fluorescence. For chemiluminescence, the protein bands can be observed using a digital imager, or with film. Fluorescence signal detection requires an imager.

How long does gel electrophoresis take?

The run time for gel electrophoresis can vary anywhere from 45 to 90 minutes. The specific time needed to run a gel depends on a variety of factors, including:

  • the degree of separation desired,
  • the voltage applied, or
  • the gel’s composition.

Gel electrophoresis steps

Gel electrophoresis is a pretty straightforward process that involves preparing the samples in loading buffer, loading the gel box with running buffer, pipetting the samples into the wells, actually running the gel, and finally, visualizing your proteins.

STEP 1: Prepare samples

Samples will differ dramatically by individual experiment, but they all must be processed similarly prior to gel electrophoresis. To begin gel electrophoresis, you will mix your samples with a loading buffer. Loading buffer contains both dye, as a visual indicator while loading and running the sample, and glycerol, to increase the density of the samples. Increasing sample density promotes sinking to the bottom of the wells during loading, preventing the otherwise light samples from quickly diffusing out of the wells during loading.

  • Quick Tip: Consider how these samples may be presented in a future figure for presentation or publication.

    For example, if a sample may need to be cut out of an image, it is advisable to load that sample on the end to prevent compromising the integrity of the image. We put together a full list of publication requirements from the most popular scientific journals, like nature, PLOS, and MDPI to help you out.

STEP 2: Prepare gel and buffer

Gels can be purchased already made (pre-cast) or they can be made in the lab (hand cast).  In preparing the gel, there are a number of factors to consider, including the gel composition, the percentage of the gel (this will affect pore size and thus separation resolution), the number of wells needed, and the size of those wells.

  • Quick Tip: Buying pre-cast gels for can save time and ensure consistency of results by removing the inherent variation that comes with making gels by hand in the lab.

Choose the type of running buffer you will need and prepare it ahead of time. Buffers can often be purchased, though these are often made in bulk in the lab. When you’re ready to load, remove the comb from the gel. Fill the gel box with the running buffer and place the gel into the box so that it is covered by the running buffer.

STEP 3: Load and pipette samples

Before loading the samples, decide on the ideal order of the samples on the gel. Using a pipette, carefully add samples to individual wells in the gel. Additionally, a ladder with specific size markers needs to be added to one of the wells as a reference for downstream analysis.

Samples loaded into individual wells in the gel.
Visual showing DNA samples loaded into the gel wells. To load the samples into the wells, you will use a pipette and pipette tips. Later, an electric current will be applied to pull the DNA through the gel. (Created with BioRender.com)

STEP 4: Electrophoresis (Run the gel)

Once the samples are loaded, place the lid on the gel box, plug the cords into the power supply, and run the gel with electrophoresis. The voltage and time required will need to be adjusted based on each lab’s specific experiment.

Representation showing the two main methods of gel electrophoresis: agarose (horizontal) or polyacrylamide (vertical).
Visual representation showing the two main methods of gel electrophoresis: agarose/horizontal (left) or polyacrylamide/vertical (right). Each type requires the same key components: a gel with wells for the sample, buffer, and an electric current. Once samples are loaded into the wells of the gel, the power source supplies an electric current that moves the molecules (DNA or protein) through the gel. (Created with BioRender.com)

STEP 5: Visualize and document bands

Azure chemisolo next to a hand using a mobile device to connect
A unique web browser interface allows the chemiSOLO to be controlled by phone, tablet, or PC, without the need to install any additional software.

When the steps for gel electrophoresis is complete, the resulting bands of DNA, RNA, or protein need to be visualized. For DNA gels, a DNA stain added to the gel allows visualization when placed under UV light. DNA and RNA blots require additional steps prior to visualization. Proteins can be visualized in the gel (such as with two-dimensional difference gel electrophoresis or 2D- DIGE); however, more often, they need to be transferred from the gel to a membrane for further analysis. Digital imagers, like the Azure Imagers, allow for both the visualization and documentation of results in one, swift step.

Azure Biosystems offers a range of imagers capable of imaging gels stained with Coomassie, silver stain, and more. Imagers able to image under white light (epi or trans-illumination) include the new chemiSOLOIt’s the first personal Western blot imager of its kind on the market! This personal Western blot imager is able to easily and quickly image chemiluminescent Western blots without additional software downloads.

Stained gels and blots can be imaged on both laser- and CCD-based fluorescent imaging systems using total protein stains, like AzureRed (shown below) or Azure TotalStain Q. AzureRed is a quantitative, fluorescent protein stain for total protein normalization in blots and total protein detection in gels. It is fully compatible with downstream Western blotting or mass spectrometry. Azure TotalStain Q can be used to see all proteins on the membrane.

Overlay of four channels. Western blot stained with total protein stain, AzureRed, probed for three proteins of interest without a destaining step, scanned with Azure Sapphire Biomolecular Imager
AzureRed is imaged simultaneously with three proteins of interest. The gel was loaded with dilutions of HeLa cell lysate. After transfer, the blot was stained with AzureRed and then probed for tubulin, ß-actin, and GAPDH without a destaining step. The blot was scanned with each of the four lasers of the Sapphire Biomolecular Imager. In this overlay of the four channels, total protein (AzureRed stain) is shown in gray; tubulin in red, ß-actin in blue, and GAPDH in green.

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Different types of gel electrophoresis

Most gels used for electrophoresis are made from either agarose or polyacrylamide. DNA and RNA are separated horizontally via agarose gels, while proteins are separated vertically using polyacrylamide gels.

Longer run times are required for higher degrees of separation. While using a higher voltage can reduce the run time, if the voltage is too high, the gel can start to melt and create fuzzy or irregular bands. Gel composition affects how much voltage can be applied; a higher voltage may cause a low percentage gel to melt, where a higher percentage gel could withstand the higher voltage.

Check out these troubleshooting resources for gel electrophoresis and SDS-PAGE

SOURCES

  1. Electrophoresis. (2022, December 8). National Human Genome Research Institute. Retrieved December 15, 2022, from https://www.genome.gov/genetics-glossary/Electrophoresis
  2. DNA Gel Electrophoresis Equipment. (2019, September 11). LabXchange. Retrieved December 15, 2022, from https://www.labxchange.org/library/pathway/lx-pathway:33b08759-5d13-4128-8867-68428a8d1081/items/lx-pb:33b08759-5d13-4128-8867-68428a8d1081:html:ca030dca?source=%2Flibrary%2Fclusters%2Flx-cluster%3Aabe

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