Polyacrylamide gel electrophoresis (PAGE) is a fundamental molecular biology technique used to separate proteins or nucleic acids based on size, shape, and charge. The process uses a porous, three-dimensional matrix, created by polymerizing acrylamide monomers with the crosslinker N,N’-methylenebisacrylamide, which acts as a molecular sieve. An electric field forces charged macromolecules to migrate through this gel; smaller molecules move faster while larger ones are retarded. Preparing this matrix requires precise measurement of reagents and strict adherence to safety protocols.
Essential Safety Protocols and Equipment Setup
Preparing polyacrylamide gels involves handling unpolymerized acrylamide, a potent neurotoxin and suspected carcinogen. Therefore, all solution preparation and gel casting must occur within a functioning chemical fume hood to prevent inhalation of airborne powder or vaporized solutions. Personnel must wear appropriate personal protective equipment (PPE), including a fully buttoned laboratory coat, chemical-resistant gloves (e.g., nitrile), and eye protection. Contaminated gloves should be changed immediately, and hands must be washed thoroughly after handling the solution.
The physical setup uses a gel casting apparatus, typically consisting of two clean glass plates separated by defined spacers (often 0.75 mm or 1.5 mm). These plates must be meticulously cleaned and assembled within the casting stand to form a leak-proof seal at the bottom. A successful seal is critical, as the liquid gel mixture will leak if the plates are not clamped tightly. Ensuring the plates are perfectly aligned and secured provides the mold for pouring the two distinct gel layers.
Calculating Reagent Concentrations and Preparing Stock Solutions
Creating a functional polyacrylamide gel requires carefully mixing four main components: the acrylamide/bis-acrylamide mixture, a Tris buffer, a denaturing agent (if performing SDS-PAGE), and polymerization initiators. The primary structural component is the acrylamide monomer, cross-linked by bis-acrylamide to form the net-like gel matrix. The concentration of total acrylamide, expressed as a percentage (%T), is the greatest factor determining the final gel’s pore size.
Higher percentage gels (e.g., 15% or 20%) have smaller pores, optimal for separating low molecular weight proteins (typically under 20 kDa). Conversely, lower percentage gels (e.g., 5% or 8%) possess larger pores and are better suited for resolving high molecular weight proteins. The ratio of acrylamide to bis-acrylamide, known as %C, is typically maintained at a standard 30:0.8 ratio to ensure appropriate cross-linking density.
The Tris-HCl buffer maintains a specific pH environment, which differs between the two gel layers. The resolving gel, where separation occurs, uses a higher concentration buffer (typically 1.5 M Tris-HCl) adjusted to a basic pH of 8.8. The stacking gel, positioned on top, uses a lower concentration buffer (usually 0.5 M Tris-HCl) set to a more neutral pH of 6.8. This pH difference is fundamental to the stacking effect, which concentrates the protein sample into a narrow band before separation begins.
The polymerization reaction is initiated by two specific reagents: ammonium persulfate (APS) and N,N,N′,N′-tetramethylethylenediamine (TEMED). APS acts as a source of free radicals, often prepared as a fresh 10% stock solution. TEMED serves as the accelerator, catalyzing the decomposition of APS to produce the free radicals necessary to start acrylamide polymerization. Since the reaction begins immediately upon addition, APS and TEMED are always the last two components added to the gel mixture, allowing time for pouring.
The Step-by-Step Gel Casting Procedure
The casting process begins with preparing the resolving gel, the lower section where protein separation occurs. Appropriate volumes of water, Tris buffer (pH 8.8), acrylamide stock solution, and necessary additives like SDS are measured and mixed thoroughly. The final polymerization reagents, APS and TEMED, are then added, mixed gently, and the solution is immediately poured into the assembled glass plate sandwich. The resolving gel must be poured quickly to a precise height, leaving space for the stacking gel and the sample comb.
Pouring must be done slowly and steadily to avoid air bubbles, which disrupt the matrix and distort protein bands. Once the resolving gel is in place, a layer of water or isopropanol is carefully layered on top using a pipette. This overlay serves two purposes: it excludes oxygen, which inhibits polymerization, and it creates a perfectly flat interface for the subsequent stacking gel. The resolving gel must polymerize completely, typically taking 30 to 60 minutes, confirmed by observing a clear, solid matrix.
After the resolving gel has fully solidified, the water or isopropanol overlay is completely removed by carefully decanting and blotting the surface with filter paper. The stacking gel solution is prepared next, using the lower concentration Tris buffer (pH 6.8) and a significantly lower percentage of acrylamide (usually 4% or 5%). As before, the APS and TEMED are added last, and the mixture is gently combined.
The stacking gel solution is then poured directly onto the resolved layer, filling the remaining space between the glass plates. Immediately after pouring, the plastic sample comb is inserted into the liquid stacking gel, ensuring it is straight and fully submerged. The comb’s teeth form the wells where protein samples will be loaded for electrophoresis. The stacking gel polymerizes for about 15 to 30 minutes, completing when the gel around the comb teeth is firm to the touch.
Once both layers are polymerized, the comb is carefully removed by pulling it straight up, creating the sample wells. The finished gel, still within its glass plate sandwich, can be used immediately or stored. For storage, the gel should be kept moist and cool, typically sealed in a plastic bag with running buffer or water and refrigerated for up to one week.