Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
To protein scientists who spend hours mixing, pouring and casting electrophoresis gels ‘from scratch’ for protein separation, pre-poured gels must seem like a dream come true. But are they always preferable to ‘homemade’ gels? And which type should you use? Here are some variables to bear in mind.
Precast vs. homemade
Because both types of gels have advantages, many labs use a combination of homemade and precast. Homemade gels are less expensive, can be tailored to your exact specifications and can be prepared as needed (assuming you have the ingredients). As Kazuyuki Atarashi, global program manager of research products at MBL International, says, “In some cases, researchers want to use gels unexpectedly and urgently.”
On the other hand, the convenience and time saved by not having to mix and pour a gel, and wait for it to set, are attractive, and so is minimizing work with toxic gel reagents such as acrylamide. But perhaps most significantly, as precast gels are more uniform in their composition, they offer improved consistency and reproducibility in the final data—factors that can impact the bottom line, says Melissa Stolow, market development leader in protein biology at Thermo Fisher Scientific.
“One must remember that if gels have to be run multiple times because self-casting does not provide equal gel consistency and performance, the difference in time to results is impacted and the cost of that must be considered when choosing precast vs. self-cast gels,” Stolow says.
Besides time, convenience and reproducibility, precast gels also boast a longer shelf life. Homemade gels can last about a week, but precast gels can be stored for up to months before use.
Splitting the difference
Labs do have more choices than precast and homemade. Researchers can aim somewhere in between with the TGX™ FastCast™ system from Bio-Rad Laboratories or the Fisher BioReagents™ EZ-Run™ Protein Gel Solution from Thermo Fisher Scientific.
FastCast enables users to mix and pour their own gels using prepared Bio-Rad reagents that simplify the process. The resulting gels are ready for use in about a half-hour vs. perhaps an hour using traditional recipes. Additionally, making gels this way is easier and more accurate than doing it from scratch. FastCast gels also separate proteins faster than traditional homemade gels, says Kate Smith, senior product manager in the lab separations division at Bio-Rad. And they last longer, about a month, so users can prepare a large batch of gels and use as needed. “The cost of FastCast gels is closer to handcast gels than to precast gels, and the reproducibility and shelf life [are] much closer to precast,” Smith says.
Key considerations
The demand for and availability of precast gels have grown so much that making a choice can be overwhelming. Many vendors offer guidance on their web sites, but here are some key points to consider.
Size/capacity
Make sure the gel you choose will fit in your gel box or system. It’s also important to think about sample volume: How many wells will you need per gel, and how much sample will you load per well? Thermo Fisher Scientific offers specially designed wells for large samples, for instance. “The Bolt® gel system is unique in its design with a wedge well to allow for loading of higher volumes of samples, which is advantageous for dilute or precious samples that need to be separated,” says Stolow.
Gel percentages
Gel percentage (usually between 5% and 20%) determines the gel’s resolving power. “Pay attention to gel percentages,” says Smith. “This helps with maximum separation and resolution.” Smith says most companies have a migration chart that shows the separation powers of their gels at different percentages; study this to pick the best gel percentage.
If you’re unsure of which gel percentage to use, try a gradient gel, says Smith. Gradient gels, which run samples through a gel-percentage gradient, such as 8% to 16% or 4% to 20%, are a great way to find out where you need more resolving power. Once you identify the gel-percentage neighborhood in which your sample(s) hang out, you can zero in near that gel percentage for better separation and resolution. Because gradient gels are even more difficult to prepare yourself than single-percentage gels, they are also a good choice for purchasing as precast, says Smith.
Buffers and chemistry
Precast gels are available using virtually any common electrophoresis buffer. It shouldn’t be difficult to find what you need, but do make sure you choose one that fits your system.
Vendors also are coming up with proprietary chemistries that are designed to achieve particular ends, such as Thermo Fisher Scientific’s NuPAGE® gels with Bis-Tris and Tris-Acetate chemistry. “These gels use a unique buffer formulation that maintains a low operating pH during electrophoresis,” says Stolow. “This helps eliminate the ‘smiles’ and poor resolution seen with Tris-Glycine SDS-PAGE gels.” Both NuPAGE and Bolt gels are designed for high molecular weight resolution, short run times (about 35 minutes, vs. 90 minutes with typical Tris-Glycine gels) and a long shelf life (16 months), says Stolow.
Bio-Rad’s Stain-Free™ chemistry is also available for its FastCast system. Stain-Free technology lets users track the progress of a gel or Western blot in real time, without stopping to check for errors and without wasting time on staining. Stain-Free gels integrate into Bio-Rad’s V3 Workflow for faster Western blotting, quick error checking and total protein normalization.
Specialized and agarose precast gels
Most precast gels offered today are geared toward separating proteins within a standard size range. While this leaves fewer options for researchers studying proteins outside this size range, there are a growing number of choices for gels that separate very large or very small proteins. For peptides or very small proteins, check out Bio-Rad’s Tris Tricine precast gels (<10 kDa) or Thermo Fisher Scientific’s Novex® Tricine Gels (<20 kDa). For high molecular weight proteins, consider Bio-Rad’s Tris Acetate precast gels.
Nondenaturing gels are offered by many vendors for researchers working with native proteins. In contrast to conventional acrylamide gels with the denaturing detergent SDS, nondenaturing gels lacking SDS separate native proteins while also preserving their folding. Examples include nondenaturing varieties of Bio-Rad’s Mini-PROTEAN® TGX Precast Gels, GenScript’s Express™ or ExpressPlus™ PAGE Gels, Lonza’s PAGEr™ Gold Precast Gels and Thermo Fisher Scientific’s NativePAGE® Gels.
Bio-Rad’s Ready Gel® Zymogram precast gels and Thermo’s Novex Zymogram Gels include substrates for proteases (either casein or gelatin), for use in separating proteases and in-gel testing of enzymatic activity . And of course, many vendors also offer isoelectric focusing (IEF) gels for separating proteins by charge instead of molecular weight—the first dimension of two-dimensional protein electrophoresis.
The advantages of precast gels are undeniable, but will they ever replace homemade gels permanently? “The convenience factors save researchers time and provide more consistent results, so they don’t have to waste time repeating experiments and they get the data they need faster and more reliably,” says Stolow. It’s hard to turn down faster and more reliable data, but some researchers feel more secure knowing they could mix and pour their own gel if needed. Maybe a combination of precast and homemade gels is the best of both worlds.