What Is Electrophoresis Gel Used For

The Practical Reality of Agarose and Polyacrylamide Gels

Most people who ask What Is Electrophoresis Gel Used For are trying to separate DNA, RNA, or proteins by size. The gel itself is just a porous matrix that acts as a molecular sieve. You cast it into a slab, load your samples into wells, run current through it, and molecules migrate at rates determined by their charge and physical dimensions. That's the entire concept. The devil is in the preparation. It separates biomolecules. That's it. But let me walk you through what actually happens when you're standing at the bench at 11 PM because something in your PCR didn't work and you need to check the product. Agarose gels are for nucleic acids in the 100 base pair to 25 kilobase range. You dissolve agarose powder in TAE or TBE buffer, microwave it until clear, pour it into a casting tray with a comb, let it set for twenty to thirty minutes, then pull the comb out carefully. If you pull too fast the wells tear and your samples leak into the adjacent lane. I've ruined three gels doing that on a cold bench before I figured out the timing. Polyacrylamide gels handle smaller fragments and proteins. They require mixing acrylamide with bis-acrylamide, adding APS and TEMED to initiate polymerization, and dealing with acrylamide which is a neurotoxin in its liquid form. You wear gloves. You don't skip the gloves.

After polymerization you place the gel in a running chamber filled with the same buffer you used to make it. Load your samples mixed with loading dye. Run at a voltage that depends on gel concentration and desired resolution. Ten percent agarose runs fine at 100 volts for forty minutes. A ten percent polyacrylamide gel for protein might run at 120 volts constant for an hour. Stain with ethidium bromide, SYBR Safe, or Coomassie blue depending on what you're running. Image under the appropriate light. Here's something most protocols don't emphasize: the buffer matters more than you'd think. TAE has lower buffering capacity and runs hotter, which can cause your gel to warp or your bands to smear if you run too long. TBE holds pH better and gives sharper bands for nucleic acids under ten kilobases, but the phosphate builds up over time and can interfere with downstream applications like gel extraction for cloning. If you're running a long gel for large fragment separation, swap to 0.5x TBE instead of full strength to reduce heating without losing buffering entirely. I learned that the hard way when a standard 1x TBE run melted a 1% agarose gel halfway through and my bands looked like abstract art. The gel percentage determines pore size. Higher percentage means smaller pores and better resolution of small molecules. A 2% agarose gel resolves fragments between 50 and 1000 bases cleanly. A 0.7% gel is better for 5 to 10 kilobase fragments. Going too low and your large fragments run off the bottom. Going too high and your small fragments diffuse into the well because the pores are too tight for them to enter efficiently. Match the percentage to your target size range. Generic 1% gels work for everything in between but resolve nothing particularly well.

For protein work, SDS-PAGE is the standard. SDS denatures proteins and coats them with negative charge proportional to mass, so separation is purely by size. The stacking gel at low pH and low acrylamide concentration concentrates all your samples into a thin band before they enter the resolving gel. If your stacking gel isn't polymerized properly or the buffer pH is off, your lanes look like they were drawn with a shaky hand instead of clean horizontal bands. I once spent two hours troubleshooting smeared protein lanes before realizing the Tris-HCl in my stacking gel buffer had degraded. New buffer preparation fixed it immediately. Always make fresh resolving and stacking buffers. Old Tris absorbs CO2 from the air and shifts pH enough to throw off the stacking effect. Native PAGE is different. You run proteins without SDS so they retain their shape and charge. This tells you about oligomeric state and conformational changes but the results are harder to interpret because migration depends on both size and charge. Use it when you need that information. Don't use it when a simple Western blot would give you a clearer answer. Pulse-field gel electrophoresis handles very large DNA molecules above 20 kilobases. Standard electrophoresis can't resolve those because they all move through the matrix at the same reptation rate. PFGE alternates the electric field direction at intervals, giving large fragments time to reorient. It takes several hours instead of forty minutes but you can separate entire restriction maps. I used this once for bacterial strain typing and resolved fragments up to 500 kilobases. The gel had to be 1% agarose cast thick, run at a low voltage around 6 volts per centimeter, with switch times ramping from 2 seconds to 20 seconds over the course of eighteen hours. You need a specialized PFGE apparatus. Regular horizontal chambers won't do it.

A few things that go wrong regularly. Bubbles under the gel from inadequate sealing cause uneven running and distorted bands. Check your comb seating before pouring. Overloaded wells distort the electric field locally and create curved bands. Keep sample volume under ten percent of well volume. Using water instead of buffer to resuspend samples causes the DNA to diffuse out of the well before any current is applied because there's no ionic strength to keep the molecule confined. Always use TE or at least some buffer in your sample resuspension. The method fails completely for molecules that don't carry a net charge under your running conditions, like some lipid-attached proteins or highly modified nucleic acids. For those you need alternative separation techniques like size-exclusion chromatography or capillary electrophoresis. Gel electrophoresis also can't distinguish between molecules of identical size but different sequence. Two fragments that are both 500 bases will run identically whether they're identical sequences or completely different. You need sequencing or restriction digestion to tell them apart. When you're done, dispose of ethidium bromide waste properly. It's mutagenic. Toss contaminated gloves and tips into a designated hazardous waste container, not the regular trash. SYBR Safe is less hazardous but still follow your institution's disposal guidelines. Waste not, want not, but also don't make your safety officer's job harder than it needs to be.