Casting polyacrylamide gels

Cast polyacrylamide gel including stacking gel at least 24 hr but not more than 48 hr prior to use. Store the gel at room temperature until needed, being sure to protect it from dehydration (e.g., store submerged in high-purity water). 

It is also possible to cast polyacrylamide gels with a gradient in pore size. This gradient can either be linear or exponential. Pore size gradients can be achieved by continuously changing the monomer concentration whilst casting the gel. 

The most commonly used combination of chemicals to produce a polyacrylamide gel is acrylamide, bis acrylamide, buffer, ammonium persulfate, and tetramethylenediarnine (TEMED). TEMED and ammonium persulfate are catalysts to the polymerization reaction. The TEMED causes the persulfate to produce free radicals, causing polymerization. Because this is a free-radical driven reaction, the mixture of reagents must be degassed before it is used. The mixture polymerizes quickly after TEMED addition, so it should be poured into the gel-casting apparatus as quickly as possible. Once the gel is poured into a prepared form, a comb can be appHed to the top portion of the gel before polymerization occurs. This comb sets small indentations permanently into the top portion of the gel which can be used to load samples. If the comb is used, samples are then typically mixed with a heavier solution, such as glycerol, before the sample is appHed to the gel, to prevent the sample from dispersing into the reservoir buffer. 

Stellwagen, NC, Apparent Pore Size of Polyacrylamide Gels Comparison of Gels Cast and Run in Tris-acetate-EDTA and Tris-borate-EDTA Buffers, Electrophoresis 19, 1542, 1998. Stellwagen, NC Gelfi, C Righetti, PG, The Free Solution Mobility of DNA, Biopolymers 42, 687, 1997. 

Monolayer casting experiments made use of similar experimental conditions to form organized and highly compressed membranes. Such films were deposited on polyacrylamide gel by dipping the polymer through the air-water interface of the trough, with monolayer compression held constant at pressures of 30 to 40 mN.m". ... 

A set of nonamphoteric buffers that are derivatives of acrylamide were synthesized specifically for casting IPG gels.2,33 They are blended with the monomer mixtures and copolymerized into polyacrylamide gel matrices. In properly oriented electric fields, IPG gels develop pH gradients that increase in pH value from the anode to the cathode. 

Most important, any methods used to produce polyacrylamide gels must be followed exactly each time they are cast, since reproducible electrophoretic separations require uniform gel-forming conditions. Current literature contains many electrophoresis procedures and applications for polyacrylamide gels. Most of these employ the polyacrylamide gel in some sort of slab format. One of the most commonly used of these procedures is discussed below. 

Reagents and supplies for SDS-PAGE (see Experiment 4) Glass or plastic plates for casting SDS-polyacrylamide gel with a 10-well comb Plastic wrap... 

Both agarose and polyacrylamide gels are solid but porous matrices, which look and feel like clear jelly (Jell-0). These gels are prepared in different ways but both take the form of a slab (or alternatively column), cast like a jelly in a mould. While agarose gels are relatively easy to prepare, polyacrylamide gels are formed by complex polymerization and chemical cross-linking, and as such are usually purchased pre-cast. 

It is nowadays more usual to carry out electrophoresis using vertical or horizontal flatbed apparatus. Polyacrylamide gels are cast in cassettes formed by two glass plates separated by spacers of the required thickness placed at the sides and bottom of the plates. The plates are clamped firmly together, and if necessary the cassette is... 

Carbonic anhydrase and transferrin were purchased from Sigma Chemical Co. (St. Louis, MO). Formic acid. Cyanogen bromide, and 3-cyclohexylamino-1-propanesulfonic acid (CAPS) were purchased from Aldrich (Milwaukee, WI). Pre-cast 10-20% gradient Tris-tricine polyacrylamide gels were purchased from... 

For 2-3 decades, research biochemists cast their own polyacrylamide gels and handled the material on a regular basis. 

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