Acrylamide Polymerization A Practical Approach ( of smaller pore-size gels than would b...)

Acrylamide Polymerization A Practical Approach

of smaller pore-size gels than would be formed in their absence (urea is often a component of gel systems used to separate small proteins and peptides). This may be due to the disruption of hydrogen bonds between monomer molecules during polymerization. Smaller pore size may also be achieved at higher polymerization temperatures, an effect also attributed to hydrogen bond disruption. Contaminants of gel additives can affect polymerization. Nonionic additives such as urea, formamide, and Triton X-100 can be deionized with a mixed-bed ion exchange resin. Use 10 gm Bio-Rad Ag 5O1 X-8 resin per 100 ml additive solution and let sit overnight. However, removal of nonionic contaminants from nonionic reagents is not practical. Therefore, all additives should be qualityassured for electrophoresis.

Time
Although visible gelation occurs in 1520 min for chemical polymerization and 3060 min for photochemical polymerization, polymerization continues much longer (see Figure 1). Ammonium persulfate/TEMED-initiated reactions should be allowed to proceed for 2 hr to ensure maximum reproducibility in gel pore size. Photochemical polymerization (riboflavin-based initiator system) usually proceeds more slowly than chemical polymerization, and is also dependent on light intensity (Shi and Jackowski 1998). However, riboflavin is usually used for polymerization of electrofocusing gels in which separation is based on charge, and for which gel porosity is of secondary importance. Thus these gels can be used shortly after visible gelation without being affected by slight variations in porosity.

Monomer Concentration
The practical range for monomer concentration is between 3%T and 30%T,
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