How Gel Electrophoresis Works & How We Save You Time & Money

Agarose gels are made from the natural polysaccharide polymers found in seaweed. Agar is extracted by heating seaweed for several hours. After washing and alkaline pre-treatment, the seaweed is submerged in boiling water.

The agar dissolves into the water and the mixture is filtered to remove any residual seaweed matter. It is easily handled and cast compared to other matrices because the casting is done physically rather than with the assistance of chemicals. It is also capable of being stored for later use.

Because agarose gel does not have a uniform pore size, it is ideal for testing that involves molecules that are larger than 200 kDa, or 200 kilodaltons. The percent of agarose in the gel influences the distances between DNA bands of different lengths.

Precast agarose gels are also available, and do away with messy and time-consuming prep work. They run up to twice as fast as traditional handcast gels and come in a variety of agarose percentages, well formats, and throughput capacities.

While most modern techniques involve agarose gel, polyacrylamide gel can also be used for specific applications in similar ways. The uniformity of polyacrylamide gel allows for better accuracy, and is controlled using acrylamide and bis-acrylamide powder to create the gel.

Varying the percentage of polyacrylamide in the gel affects the pore size. Because polyacrylamide gel is biologically inert, modulating the stiffness of the gel using the powder does not affect its biochemical properties. This allows for observations that can be made based on the stiffness of the gel and no other competing changes in cellular functions.

This technique is used to separate and analyze DNA or RNA fragments by size and reactivity. It involves segmenting the molecules using a restriction enzyme.

After segmentation, the samples are placed in the gel for size and charge separation, and are then dyed using a fluorescent dye specifically for DNA. This allows for easy observation. Agarose gel is most often used in systems that perform nucleic acid gel electrophoresis.

Some applications of agarose gel electrophoresis include:

• Analysis of PCR (polymerase chain reaction) products. Examples include genetic fingerprinting, molecular cloning, or molecular genetic diagnosis
• Separation of restricted genomic DNA before Southern transfer, or Southern blot
• Separation of DNA fragments for extraction and purification
• Estimation of the size of DNA after digestion using a restriction enzyme

There are a variety of nucleic acid-specific devices available, including vertical electrophoresis systems, which are available in midid or mini gel electrophoresis format. Vertical systems are more ideal for proteins, unlike horizontal systems which are well suited for DNA and RNA.

The vertical systems utilize a discontinuous buffer system, where the top chamber contains the cathode and the bottom chamber contains the anode, and a thin gel is poured between two glass plates that are mounted. This submerges the bottom of the gel in one chamber, while the top of the gel is submerged in buffer in the top chamber.

After applying an electrical current, a small amount of buffer migrates through the gel from the top chamber to the bottom chamber. This allows for precise control of voltage gradients during separation, and results in greater separation and resolution.

Polyacrylamide gel is often used in gel electrophoresis of proteins, and is ideal for protein separation and analysis. This method is commonly used in the field of immunology. It is typical for polyacrylamide gel electrophoresis (PAGE) systems to run vertically, so that the stacking and resolving gels form a continuous gel, which would be much more difficult in a horizontal gel.

It also allows a much greater protein amount to be loaded onto the gel. Protein Electrophoresis analyzes the proteins in a fluid or extract. Because of practical limitations, protein electrophoresis is generally not suited as a preparative method.

PAGE applications include:

• Peptide mapping
• Estimation of protein size
• Protein quantitation
• Measuring molecular weight
• Comparison of the polypeptide composition of different samples
• Analysis of the number and size of polypeptide subunits

Buffers are used to help transmit the electrical charge through the gel, and maintain a stable pH level to minimize any change that may occur. The buffer may also help with the extraction of DNA.

There are a number of buffers used for agarose electrophoresis, some of which include TAE and TBE. TAE contains a mixture of Tris base, acetic acid, and EDTA (Ethylenediaminetetraacetic acid), while TBE contains a mixture of Tris base, boric acid, and EDTA.

These buffers contain EDTA to inactivate many nucleases which require divalent cation for their function, providing good conductivity while producing less heat.

The power supply of your gel electrophoresis equipment can be just as important as your gels and buffers. There are various electrophoresis power supplies available for you to use, including

Automated systems are an easy-to-use alternative to traditional gel electrophoresis instruments that provide an increase in throughput and a decrease in turnaround time.

Automated electrophoresis is best suited for a lab that wants to perform repetitive analyses, wishes to cut down on high labor cost, and has small amounts of precious samples to work with. The speed and convenience offered by an automated system that includes digital data storage for any recording is an important advantage to consider.

They incorporate DNA microfluidic kits and Chip (chromatin immunoprecipitation assay) systems for separation, sizing, and quantification of nucleic acids and proteins. The process is simplified so that minimal volumes of sample are required and results are produced in less than an hour, in contrast to manual processes that typically require substantial amounts of a sample and take over an hour to produce any results.

However, it should also be noted that being able to perform both manual and automated electrophoresis is a realistic approach for almost any laboratory, because each method offers specific advantages.

There are two different types of gel conditions: denaturing and native. Denaturing gels operate under conditions that disrupt the natural structure of an analyte, which causes it to unfold into a linear chain. Because of this, the mobility of a macromolecule only depends on its length and mass-to-charge ratio. Because the biomolecular structure is disrupted, the only structure left to be analyzed is the primary.

Native gels perform in non-denaturing conditions so that the analyte’s natural structure remains intact. This allows for the molecule’s physical size to affect its mobility, and be more easily analyzed on all four levels of its biological structure. The separated molecules in a native gel differ in molecular weight and charge, but also experience different electrophoretic forces based on its overall shape.