Gel Electrophoresis - a Crash Course
My first article will describe the purpose and design of agarose gels used in nucleic acid electrophoresis protocols. My second article will cover polyacrylamide gel electrophoresis used in protein separation, especially in Western Blot assays.
Agarose gels are employed in nucleic acid electrophoresis, i.e separating fragments of DNA or RNA on the basis of their size. Agarose is a polysaccharide(starch) derived from seaweed. When mixed with a solvent such as TAE and heated (a microwave oven works well) powdered agarose will dissolve readily then form a gel (sort of like jello) as the solution cools.
In order to load and visualize the DNA, two additional steps are necessary after the gel is cast. First, ethidium bromide (EtBr), which fluoresces orange when exposed to ultraviolet light, can be added directly to the liquid gel or to the surrounding buffer one to two hours prior to running the gel. etBr is an intercalating agent that inserts itself between base pairs of DNA, allowing each band to be visualized. Second, a comb is placed at one end of the gel prior to polymerzation in order to create wells in which to load the DNA samples.
After the gel is cast and submerged in a saline based buffer, the rest of the procedure is straightforward. As a general rule, DNA samples are mixed with a loading buffer containing glycerol to weigh down the sample and a dye such as bromophenol blue which helps visualize the dye front once electrophoresis has begun. DNA samples are then loaded carefully into each well. The extreme left or right lane of the gel is reserved for DNA standards - fragments of DNA of known base pair lengths that serve as a standard of comparison with the actual DNA. The last thing to remember is that electrophoresis of DNA occurs from the negative electrode toward the positive electrode. These are usually color coded black and red respectively in order to avoid confusion.
The voltage used to run an agarose gel ranges from 100-150V. Voltages in excess of 150V may cause the gel apparatus to overheat and cause the gel to disintegrate (esp. low percentage agarose gels). In any case, in 30 minutes or less, the bands of DNA should have separated enough to yield a basic idea of the size composition of the sample. Remember, large bands will stay towards the top of the gel whereas smaller bands will travel farther towards the positive end in the same span of time.
After electrophoresis is complete, a photograph is usually taken of the gel for purposes of documentation. At this point, the gel may be discarded or its contents may be used for other assays such as DNA/RNA extraction and purification, DNA sequencing, Southern Blot(DNA hybridization), or Northern Blot(RNA hybridization).
Western Blots and PAGE
After covering agarose gels in the first section of my article, here's a crash course in polyacrylamide gel electrophoresis (PAGE). PAGE is an essential tool in protein separation, esp. for a technique known as Western Blot, which will be discussed shortly. Similar to agarose gel electrophoresis, the basis of protein separation in PAGE is size - larger proteins move through a gel more slowly than do lower molecular weight proteins. This rule applies generally, but remember - proteins that are heavily phosphorylated or glycosylated will move differently than unmodified proteins of the same molecular weight.
PAGE is the first step in a Western Blot, a technique whose ultimate goal is the identification of a specific protein band within a sample containing a multitude of proteins, e.g. a whole cell lysate. The gel set up in PAGE, however, is more elaborate than in agarose gel electrophoresis. First the separating gel is cast. This gel consists of water, bis-acrylamide, the buffer Tris (to control pH), sodium dodecyl sulfate (SDS)(which swamps the native charge on each protein), ammonium persulfate (APS), and the polymerizing reagent TEMED. The percentage of acrylamide in these gels ranges from 8-15% of the final concentration. Lower percentage gels are more fragilebut allow for a faster separation time; higher percentage gels are more sturdy and often provide better band resolution.
Casting the separating gel. Briefly, each reagent is added except for TEMED. Once the gel apparatus is set up vertically and tested for leaks, TEMED can be added to the mixture, which is then mixed and pipetted between the two glass plates. A few minutes after pipetting the gel mixture, a layer of water or butane should be carefully added on top of the gel to prevent drying. Polymerization takes approximately 30 minutes.
Preparing the stacking gel. This gel will be added on top of the separating gel and contain the wells into which the protein sample will be loaded. The stacking gel contains the same reagents as the separating gel but with a lower percentage acrylamide. Before pouring the stacking gel, ensure that the separating gel has hardened and pour off the top layer of water/butane. Pipette the stacking gel mixture quickly, as TEMED will cause rapid polymerization. Insert a comb (most contain ten wells between the glass plates and wait 10-15 minutes.
The protein samples used in Western Blot are generally mixed with Lemmle buffer (which contains the blue dye bromophenol blue) and a denaturing agent such as DTT (dithiothreitol) or BME (beta-mercaptoethanol). Samples are boiled for 3-5 minutes prior to loading. Molecular weight markers containing multicolored standards are added to the extreme left or right lanes on the gel. Most gels are run at 70-150V in a running buffer that consists of SDS, Tris, and glycine. Please consult a protocol guide for detailed running buffer recipes.
The Transfer. After one to two hours, PAGE is complete and the gel is ready to be transferred to a blotting membrane. The most commonly used material is PVDF (polyvinyldifluoride); other membranes still in use include nylon and nitrocellulose. The idea is to transfer proteins from the gel to the membrane while preserving the exact separation pattern obtained in the PAGE step. Transfer involves a transfer buffer containing Tris, glycine, and methanol. Transfer cells work well at 100V for 60 minutes or 25-30V overnight, preferably refrigerated.
The Western blot itself is relatively simple. Once the transfer cell is disassembled and proper transfer of the proteins from the gel to the blotting membrane has been confirmed, the blot is dipped in methanol and allowed to dry. Blots can be stored for weeks to months at -20C if desired. To complete the Western Blot, the membrane is blocked in a solution of 5% non-fat dry milk in TBS-Tween for at least 60 minutes. Next comes an incubation with the primary antibody for at least 60 minutes (or overnight with shaking in a refrigerator). After three washes with TBS-tween, the secondary antibody is applied. This antibody recognizes the primary antibody and contains a detection mechanism such as a fluorescent tag, the enzyme HRP (horseradish peroxidase), or a radioisotope such as iodine 125. After three more washes, the blot can be developed and visualized with X-ray film in a dark room.
After the development step, the blot may either be discarded or stripped and reprobed several times for other proteins.
Best of luck and happy blotting!