Electrophoresis involves separating and analyzing charged molecules, mainly proteins or nucleic acids, using an electric field. In gel electrophoresis, the separation of molecules being analyzed takes place in a gel and is based on differences in their size, charge, conformation, and few other factors. Similar to other analytical techniques, recent advances in gel electrophoresis have all focused on improving speed, accuracy, automation, and reducing the cost of analysis through changes in protocols for gel preparation, sample separation, sample visualization, and analysis.
Eco-friendly sample preparation
Sample preparation for gel electrophoresis involves isolating the DNA, RNA, or protein from the cell or tissue sample using PCR or enzymatic digestion. The mobility of an analyte through the gel is determined by the current and voltage used, the buffer composition, the gel concentration, and the temperature used. The higher the concentration of the gel, the denser is the sieve created to separate small fragments. The secondary structure of a protein or RNA also determines how fast it migrates through the gel, and this structure can be minimized or eliminated using a reducing agent that breaks the bonds and disrupts the conformation within the molecules. Once denatured, the linear protein or RNA migrates across the gel proportional to its size. Native gel electrophoresis keeps the protein or RNA in its native confirmation, while denaturing gel electrophoresis disrupts the conformation and reduces the molecule into a linear structure before or during electrophoresis. Polyacrylamide gel electrophoresis (PAGE) is used to separate proteins and small DNA fragments, whereas denaturing PAGE is commonly used for separating RNA. Agarose gels are typically used to separate DNA fragments ranging from few hundred to about 20,000 base pairs.
In DNA analysis, once the samples have migrated through the gel, the gel is placed into an ethidium bromide (EtBr) dye solution. EtBr helps in sample visualization as it intercalates between DNA and is visible upon UV light excitation. The DNA bands in the sample and the reference lanes can be estimated and excised easily from the gel using this staining method. EtBr dye is affordable and fairly sensitive, which is why it has been used for nucleic acid staining for decades. However, it is also highly mutagenic and the costs associated with decontamination and disposal of this dye are not insignificant. Some alternatives are available but they are not ideal either, as those dyes have issues with sensitivity, stability, and cytotoxicity.
“We believe that a DNA-binding dye can be made non-mutagenic by denying its chance to be in contact with genomic DNA in living cells,” says James Maclean, Ph.D., Product and Business Development Manager at Biotium. “We engineered the chemical structures of GelRed® and GelGreen® such that the dyes are incapable of crossing cell membranes of living cells. Furthermore, environmental safety tests showed that these dyes are non-hazardous and non-toxic to aquatic life. As a result, they can be disposed of down the drain or thrown away in the regular trash.”
Multiplexing and automation
The use of gel electrophoresis has always been limited by its throughput and automation capabilities. Based on feedback that products on the market do not provide the accuracy and control automation required for many applications, LabSmith recently launched uEP01-300 and uEP300-12 to fill a need for small size, easily multiplexed, fully automatable gel electrophoresis power supplies. “We started the development as a custom product for a long-time customer who had a 12-channel gel electrophoresis system and didn’t want to stack a dozen of the off-the-shelf power supplies together,” says Kristen Pace, CEO at LabSmith. “With our uEP01-300 system, each channel can be run at constant voltage, constant current, or constant power with 16-bit accuracy. Our software makes it easy to create multi-step automated scripts, and to log the voltage and current measurements. This is a very powerful capability for researchers who need a well-controlled and documented process flow.”
Image: The LabSmith uEP01-300 is a compact, breadboard-based 300V power supply for electrophoresis and gel electrophoresis setups.
Reagents and kits for gel electrophoresis are also being developed with the goal of improving multiplexing. “When performing a multiplex experiment combining multiple antibodies, background noise becomes a critical issue,” says Ben Wang, Ph.D., Product Manager, Protein Quantitation at Bio-Rad Laboratories. “We recently launched a new 5-minute blocking buffer called EveryBlot that is formulated to give the user quality blots with clean signal.”
Besides multiplexing, ease of integration, software customization, and increasing the compactness of the instrument are also playing a key role in guiding the development of next-generation gel electrophoresis systems. Some electrophoresis systems now come packaged with all of the equipment—gel tank, power supply with pre-set voltages, keypad and a timer, and casting set (tray, casting stand, tooth combs)—and reagents needed to run the experiment. They also include built-in features to save time for gel casting and sample loading and for better visualization of wells during sample loading and to track the migration across the gel. They improve experimental safety by allowing for heat dissipation and letting the current flow only when the equipment is properly set-up.
“The next step is the automation of electrophoresis to limit personnel contact during sample handling,” says Wang. “We will start to see more systems that can perform the entire workflow just by providing sample input.” The use of pre-prepared and pre-packaged gels and reagents are the first step toward this goal as it minimizes the steps for sample preparation and handling.
“Time is our most valuable asset and simple kits that produce reliable results are now widely available,” says Shea Biondi, Director of Marketing at Zymo Research Corporation. Zymo Research’s website offers information on the Zymoclean Gel DNA Recovery Kit and includes many best practices for ensuring maximum DNA recovery from the gel. “These days it is very easy to achieve high purity concentrated DNA from a gel slice for well under $2 per preparation, that is immediately ready for DNA ligation, sequencing, labeling, PCR, NGS, and other applications.”
Gel electrophoresis is a very saturated market with a lot of products available for use. “There are many critical steps in the electrophoresis workflow and anything you can to do shorten the process and provide consistency will be very beneficial,” says Wang. “Select something that will give you consistent results and cut your time to results without compromising either. This will ultimately help you achieve your experimental goals.”