Electrophoresis Gel Selection

by K Chico

Gel electrophoresis is a technique that uses electrical current to separate DNA, RNA or other proteins based on their size, shape, and charge. Gel construction is simple, quick, and relatively inexpensive and finished gels can have the consistency of a firm Jello to a rubberier substance, depending on concentration. Additionally, certain parameters of gels, like concentration and dimension, can easily be tuned depending on experimental need. Historically, starch gels containing potato starch have been used for zone electrophoresis, and more recently, research on corn starch as a gel medium has been conducted. Even so, agarose and polyacrylamide gels remain the gold standards.

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
Disdvantages	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

Agarose Gel

Comparison of DNA detection in 1% agarose gel in TBE buffer using Gelite™ Safe and GelRed®. 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 and GelRed™ (Biotium) 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 an EtBr filter set.

Agarose is a natural linear polysaccharide that can be isolated from some seaweeds. An agarose gel is prepared by pouring the warm liquid agarose solution into a casting tray with a comb that molds the wells for each sample. Upon casting, these sugars become interlinked to form pores, and the gel will set as it cools. Agarose gels typically utilize a horizontal apparatus where dimensions can be easily modified. A larger gel, for example, allows samples to run on the gel for longer durations without running off into the buffer. The horizontal setup additionally makes this technique suitable for immune electrophoresis. Recently, a vertical system combined with sodium dodecyl sulfate (SDS) has been developed to separate very large proteins (200-4,000 kDa).

Although agarose gels are non-toxic and easy to handle, they do not exhibit great uniformity in pore size, which becomes smaller with increasing agarose concentration. Likewise, agarose gels can only separate double-stranded (ds) DNA; though they maintain their status as matrix of choice for the separation of nucleic acids since polyacrylamide gels can be toxic in the non-polymerized form and complex to prepare. Agarose gels have a lower resolving power than polyacrylamide gels but have a greater range of separation. Agarose gels are generally suitable for separating molecules from roughly 50 bp-20 kbp, which makes them increasingly useful for large DNA fragments, such as the products of PCR. In some techniques like pulsed field gel electrophoresis, resolution of over 6 Mb is also possible using agarose gels.

Polyacrylamide Gel

Polyacrylamide gels are created by the polymerization of acrylamide monomers in the presence of bisacrylamide, a crosslinking agent. Polyacrylamide gels are created by either pouring the warm polyacrylamide solution in the space between two parallel vertical glass plates, or by using a glass cassette. The chemical crosslinking of acrylamide and bisacrylamide creates a sieved membrane in which fragments become trapped. Polyacrylamide gels typically utilize a vertical apparatus, where a comb is placed on top of the gel to create each sample well, and samples run downward instead of horizontally.

The pore size of polyacrylamide gels is incredibly consistent and reproducible and is directly related to the ratio of acrylamide to bisacrylamide (which inherently determines the concentration of the gel). Though construction of these gels is more expensive in time and resources, they often provide more reproducible results over agarose counterparts. Polyacrylamide gels can also separate DNA in ds and single strand (ss) form and can be further modified to acquire superior resolution for nucleic acids. For example, denaturing polyacrylamide gel can distinguish ssDNA molecules that are only one nucleotide different, allowing for subsequent Sanger sequencing.

Polyacrylamide gels are widely used for small nucleic acids (e.g., miRNA and tRNA) as well as other proteins because they are more adaptable to accommodate several aspects of protein characterization. In native polyacrylamide gel electrophoresis (PAGE), proteins can be distinguished based on the natural or denatured confirmation using non-denatured gels. Alternatively, proteins can also be separated based on their size instead, when using higher percentage (10%-20%) gels with SDS, a denaturing agent.

Products

Table 2. 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

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Table 3. Specifications for fluorescent protein gel stains.

Stain ▲ ▼	Ex/Em (nm) ▲ ▼	Detection Range ▲ ▼	Stain Time ▲ ▼	Applications ▲ ▼	Fixatives Requried ▲ ▼	De-staining Required ▲ ▼	Cat No. ▲ ▼
ProLite™ Orange	484/586 nm	0.8 ng to 15 µg	∼1 hour	1D SDS-PAGE	No	No	18000 (100 µL) 18001 (1 mL)

References

Manual for Starch Gel Electrophoresis: A Method for the Detection of Genetic Variation
Assessment of corn starch as substitute for agarose in DNA gel electrophoresis
Gel Electrophoresis
Principles and Techniques of Biochemistry and Molecular Biology Seventh edition

Original created on August 28, 2023, last updated on August 28, 2023
Tagged under: electrophoresis, gel, agarose, polyacrymalide, DNA, RNA, protein

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