Gel Electrophoresis

Gel electrophoresis is a core molecular technique used routinely in life science research to separate and analyze charged macromolecules. By applying an electrical field to a gel-based medium, macromolecules such as proteins and nucleic acid fragments can be separated and purified according to their size, charge, or conformation. Although commonly performed as an analytical procedure ensuing PCR, gel electrophoretic methods have also found much success as a preparative technique before blotting, cloning, DNA sequencing, and other various downstream applications.

Principle of Gel Electrophoresis

As mentioned previously, gel electrophoresis involves using an electrical field to separate proteins and nucleic acid fragments based on size, charge, or conformation. This field is typically generated in an electrophoretic chamber (e.g., a hard plastic tank), commonly known as a gel box (Figure 1).

Figure 1. An illustration of gel electrophoresis workflow (figure made in BioRender).

A gel box is a buffer-filled container configured with a cathode (negative terminal) at one end, an anode (positive terminal) at the opposite end, and an external power source. Gels, which behave as both an anticonvective and sieving medium, are first submerged in the buffer with its wells near the anode. Then using a pipette, samples are loaded into the wells of the gel. When the external power source is turned on, an electrical field is applied to the buffer causing charged molecules to move through the gel towards the opposite terminal (i.e., towards the anode). Depending on the size of the molecule, it will travel through the matrix at different rates and distances. Smaller molecules will migrate through the gel matrix faster than larger ones, with the shortest molecules traveling the furthest.

Types of Gel Electrophoresis

The two most common gel electrophoretic methods include agarose gel electrophoresis for separating nucleic acids and polyacrylamide gel electrophoresis (PAGE) for separating proteins, and nucleic acid fragments 500 base pairs or less. The following table summarizes key differences between agarose and polyacrylamide gel electrophoresis.

 

Table 1. Summary of the key differences between agarose and polyacrylamide gel electrophoresis.

Electrophoretic Method Agarose Gel Electrophoresis Polyacrylamide Gel Electrophoresis
Gel Type Agarose Polyacrylamide
Gel Composition Agarose is a polysaccharide extracted from seaweed. The gel contains long chains of interlinked sugars to form a meshwork. Polyacrylamide is a synthetic resin made by digesting acrylonitrile with nitrile hydratase. The gel is made by the chemical crosslinking of acrylamide and bis-acrylamide to produce a molecular sieve.
Gel Casting Method Gel sets as it cools. Agarose powder is mixed in a buffer and microwaved. The mixture is then poured into a gel frame and allowed to set. Gel sets by a chemical reaction once crosslinking occurs.
Pore Properties Concentration dictates pore size. Pore size becomes smaller as the concentration increases. (Typical concentration is 0.5-2%). The ratio of acrylamide to bis-acrylamide dictates pore size. (Typical concentration is 6-15%)
Run Configuration The gel is run horizontally. The gel is run vertically.
Application Separates nucleic acid fragments 50-20,000 base pairs in length. Separates proteins and nucleic acid fragments 5-500 base pairs in length (e.g., oligonucleotides, miRNA, tRNAs, etc.).
Advantages
  • Nontoxic gel medium
  • Gels are simple to cast and set quickly
  • Good for separating large DNA molecules, such as PCR products and double-stranded DNA
  • Samples can be recovered from the gel either by melting the gel, digesting with enzymes agarose, or treating with chaotropic salts
  • Stable chemically cross-linked gel
  • Pores are uniform in size
  • High resolving power, sharp bands
  • Good for separating low molecular weight fragments such as single-stranded DNA
Disadvantages
  • Agarose is costly
  • Comparatively low resolving power, fuzzy bands
  • Poor separation of low molecular weight fragments
  • No uniform pore size
  • Toxic monomers (i.e., acrylamide is a potent neurotoxin)
  • Gels are tedious to prepare and are prone to leakage
  • Each experiment requires a new gel

Visualizing DNA Samples After Separation

After separation by electrophoresis, nucleic acid fragments can be visualized using several different methods. The most well-known method for visualizing nucleic acid fragments is staining with ethidium bromide (EtBr). However, because EtBr is highly carcinogenic, cytotoxic, and mutagenic. It is environmentally hazardous and must be handled and disposed of carefully. Safer alternatives to EtBr include novel gel stains, such as Gelite™ Safe *10,000X Water Solution*, Gelite™ Safe *10,000X DMSO Solution*, Helixyte™ Green and Helixyte™ Gold, and gel staining kits, such as Gelite™ Green Nucleic Acid Gel Staining Kit and Gelite™ Orange Nucleic Acid Gel Staining Kit. Not only are these gel stains and kits less mutagenic than EtBr, but Gelite™ Safe, Helixyte™ Green, Helixyte™ Gold, and Gelite™ nucleic acid staining kits can detect nucleic acids in both agarose and polyacrylamide gels, with greater sensitivity and less background interference.

Gelite™ Safe – the most sensitive and robust DNA gel stain

Gelite™ Safe is an extremely sensitive and stable fluorescent dye for visualizing DNA in electrophoretic gels. Unlike the highly-toxic EtBr, Gelite™ Safe is explicitly designed to be less hazardous, non-cytotoxic, and non-mutagenic, providing greater sensitivity and lower background fluorescence than EtBr and SYBR® Safe. The strong DNA binding affinity of Gelite™ Safe allows for DNA to be stained before or post electrophoresis without destaining, and because Gelite™ Safe is specific to DNA, applications in which RNA is present in the sample will not obscure results. Gelite™ Safe has fluorescence excitation maxima at 280 nm and 513 nm and a broad emission profile with a maximum at 552 nm. These unique spectral properties broaden the instrument compatibility of Gelite™ Safe to include UV and blue-light transilluminators, gel documentation systems, and laser scanners, and its single formulation design allows for detection in both the green or red channels at your convenience.

Figure 2. Comparison of DNA detection in 1% agarose gel in TBE buffer using Gelite™ Safe, EtBr, and SYBR® Safe. Two-fold serial dilutions of 1 kb DNA ladder were loaded in amounts of 100 ng, 50 ng, and 25 ng from left to right. Gels were stained for 60 minutes with Gelite™ Safe, EtBr, and SYBR® Safe according to the manufacturer’s recommended concentrations and imaged using the ChemiDoc™ Imaging System (Bio-Rad®). Gels were illuminated using a 300 nm transilluminator fitted with a GelGreen filter.

Table 1. Nucleic acid stains for agarose and polyacrylamide gel electrophoresis

Product Ex (nm)¹ Filter² Unit Size Cat No.
Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution* 254 mn Long path green filter 1 mL 17590
Helixyte™ Green Nucleic Acid Gel Stain *10,000X DMSO Solution* 254 mn Long path green filter 100 µL 17604
Helixyte™ Gold Nucleic Acid Gel Stain *10,000X DMSO Solution* 254 mn Long path green filter 1 mL 17595
Gelite™ Green Nucleic Acid Gel Staining Kit 254 nm or 300 nm Long path green filter 1 Kit 17589
Gelite™ Orange Nucleic Acid Gel Staining Kit 254 nm or 300 nm Long path green filter 1 Kit 17594
Gelite™ Safe DNA Gel Stain *10,000X Water Solution* 254 nm, 300 nm or 520 nm Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters 100 µL 17700
Gelite™ Safe DNA Gel Stain *10,000X Water Solution* 254 nm, 300 nm or 520 nm Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters 500 µL 17701
Gelite™ Safe DNA Gel Stain *10,000X Water Solution* 254 nm, 300 nm or 520 nm Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters 1 mL 17702
Gelite™ Safe DNA Gel Stain *10,000X Water Solution* 254 nm, 300 nm or 520 nm Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters 10 mL 17703
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution* 254 nm, 300 nm or 520 nm Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters 100 µL 17704
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution* 254 nm, 300 nm or 520 nm Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters 500 µL 17705
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution* 254 nm, 300 nm or 520 nm Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters 1 mL 17706
Gelite™ Safe DNA Gel Stain *10,000X DMSO Solution* 254 nm, 300 nm or 520 nm Ethidium Bromide, Gel Star, Gel Green, Gel Red and SYBR filters 10 mL 17707
  1. Excitation settings are for a transilluminator or laser-based gel scanner.
  2. Common long path green filters include the SYBR® filter and GelStar® filter.

 

Visualizing Protein Samples After Separation

For proteins, visualization can be achieved using either color-producing chemical reactions or dye-binding methods. Determining which method to use depends largely on the conditions of the experiment and the intended downstream application. Common strategies for detecting proteins in gel include Coomassie dye staining, silver staining, zinc staining, and fluorescent dye staining. While Coomassie dye and silver staining methods remain largely popular for total protein profiling, fluorescent dye staining methods offer greater sensitivity and linear dynamic range.

 

Table 3. Summary of methods for staining proteins in gel.

Stain Sensitivity Range Protocol Time Detection Method Compatible Downstream Applications Notes
Coomassie Dye 5 – 25 ng 10 – 135 minutes
  • Colorimetric (visual)
  • Mass spectrometry
  • Sequencing
  • Western blotting
  • Quick and easy staining protocol
  • Reversible
Silver stain 0.25 – 0.5 ng 30 – 120 minutes
  • Colorimetric (visual)
  • Mass spectrometry
  • Most robust and sensitive colorimetric method
  • Protocol requires several steps
  • Stain uses either glutaraldehyde or formaldehyde as an enhancer, which can cause proteins to crosslink in the gel matrix
Zinc stain 0.25 – 0.5 ng 15 minutes
  • Colorimetric (visual)
  • Mass spectrometry
  • Western blotting
  • Procedure stains the polyacrylamide gel and not the proteins
  • No fixation steps required
  • Stain can be easily removed
Fluorescent dye 0.25 – 0.5 ng 60 minutes
  • UV/blue/green-light transilluminators
  • Gel documentation systems
  • Laser scanners
  • Mass spectrometry
  • Western blotting
  • Wide linear dynamic range and sensitive
  • Compatible with destaining and protein recovery methods
  • Routinely used in 1D and 2D applications

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