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.
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 type2. What you’ll need to begin gel electrophoresis:
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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
STEP 1: Prepare the 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.
STEP 2: Prepare the 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.
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 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.
STEP 4: Electrophoresis (Running 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.
STEP 5: Visualize and document bands
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.
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Stained gels and blots can be imaged on both laser- and CCD-based fluorescent imaging systems using total protein stains, like AzureRed (shown above) 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.
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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
- Electrophoresis. (2022, December 8). National Human Genome Research Institute. Retrieved December 15, 2022, from https://www.genome.gov/genetics-glossary/Electrophoresis
- 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
