by Caitlin Smith
The central principles of protein electrophoresis have not changed in the decades since its inception. However, improvements and refinements continue to make it one of the most important techniques in the life sciences. Two-dimensional (2D) protein electrophoresis remains unrivaled as a simple, inexpensive, and accessible tool for separating proteins in a mixture. This is especially true for complex biological samples such as a tissue homogenate, from which low-abundance proteins (often of particular biological interest) can be difficult to pull out.
During electrophoresis, the force of an electrical current passing through the gel matrix causes the different protein species in the sample mixture to migrate through the gel at different rates. Differences in electrical charge, size, shape, addition of modifications (such as phosphate groups), and multimerization can all influence a protein’s rate of migration during electrophoresis. Before electrophoresis, most proteins are denatured with a detergent such as sodium dodecyl sulfate (SDS) to help the proteins relax and cover them with negative charges. When coated with SDS, proteins migrate mainly according to size, not according to charge or shape. Here is a look at some new tools to take advantage of when enjoying the many benefits of this old technique.
Precast slab gels save time
A major supplier of electrophoresis tools for decades, Bio-Rad continues to innovate with its electrophoresis line. “Bio-Rad was the first to introduce a precast gel to the market and this forever simplified and standardized the use of electrophoresis in protein research,” says Christopher Belisle, PhD, Marketing Manager, Electrophoresis & Blotting from Bio-Rad. “Bio-Rad is continuing to launch the Mini-Protean and Criterion TGX and TGX Stain-Free gels in 2010. These precast gels come in both the mini and midi formats.” The TGX gels have the fastest running times (15 min for mini, 20-25 min for midi), and the fastest transfer times for Western blotting (15-30 min). No special buffers are required as they run with Laemmli buffer. The TGX–Stain Free gels additionally allow you to run and image gels in 20-25 minutes. “No other gel in the market can run as fast or transfer proteins as efficiently while using the gold standard Laemmli buffer,” says Belisle. “The TGX Stain-Free technology is only offered by Bio-Rad and further enhances our commitment of time to results for the researcher.” Belisle believes that the major developments in protein electrophoresis will arise from improvements to the workflow that make the process more efficient. “These new [precast and stain-free] gels allow the researcher to focus on the science and get more done in less time with more reproducible results,” says Belisle.
Invitrogen also offers precast gels in their NuPAGE® and Novex® systems. In addition, their ZOOM® Benchtop Proteomics System offers an integrated solution for protein profiling. It puts together sample fractionation, 1D and 2D gel electrophoresis, and stains compatible with mass spectrometry.
Freedom from slab gels
When using SDS-PAGE to separate proteins for further analysis, such as a sample prep method prior to mass spectrometry, the tricky process of excising bands from the finished gel begins. Another way to collect proteins separated by electrophoresis is with a fractionation system. Protein Discovery’s Gelfree 8100 Fractionation System is designed to replace electrophoresis gel band and spot cutting with programmable liquid-phase fraction recovery. “Like regular slab-gel PAGE, [the Gelfree 8100 Fractionation System] fractionates complex protein mixtures by size, but the innovative aspect of the system is that it uses a horizontal tube-gel cartridge format with liquid reservoirs at each end to simplify sample loading and fraction collection,” says Matthew Wygant, director of marketing at Protein Discovery. “The only specialized skill required to get good protein electrophoresis results from the Gelfree system is good pipetting technique.” Both systems – traditional slab gels and Protein Discovery’s Gelfree system – use SDS-PAGE to separate protein mixtures according to size. However, the Gelfree system is optimized for sample preparation. “You can load so much more protein, recoveries are around 80%, and fractions are delivered directly into small liquid volumes that you can just pipette out and take directly to the next step in your workflow,” says Wygant.
From electrophoresis to mass spectrometry
Wygant believes that the most exciting recent developments in protein electrophoresis involve its use as a sample prep method for mass spectrometry. “The speed, sensitivity, and resolution of mass spectrometer instrumentation have continued to increase in recent years, and a lot of protein researchers now have access to really powerful mass spec systems,” says Wygant. “But they're finding that they still can't get a good read on the low-abundance proteins where all the interesting signaling and regulation is going on. Protein mixtures that are analyzed without some kind of pre-fractionation are so complex that the minor elements, the ones of interest, are lost among signals from the more abundant ones. But non-electrophoretic prefractionation methods seem to be not specific enough, so researchers end up with not enough signal from the mass spec to do a confident analysis.” Historically, though, using slab gel electrophoresis as a preparative system is challenging. Excising bands from gels can result in little or no protein. “There
really wasn't a reliable, high-yield way to use gel electrophoresis to prepare proteins for mass spec analysis prior to Gelfree,” says Wygant.
A future hurdle to jump in using electrophoresis as a preparatory method for mass spectrometry is in finding a replacement for detergents such as SDS. “Protein electrophoresis is almost always carried out with the aid of a surfactant, and that surfactant is almost always SDS,” notes Wygant. “In addition to its aggressive detergent properties that are useful for extraction and solubilization, SDS is, of course, also used to apply a net negative charge to proteins for size-based fractionation in SDS-PAGE. But if you're going to the mass spec, SDS will cause problems, so you have to get rid of it. There are a variety of effective protocols, and it isn't that hard, but still you have this bump in your workflow where you have to remove SDS from all your samples. In the future, I expect to see scientists working on faster ways to get rid of SDS and perhaps specialized mass spectrometry compatible reagents that can replace SDS in protein electrophoresis.”