FORMATION OF A CYSTEINE ACRYLAMIDE ADDUCT IN ISOELECTRIC-FOCUSING GELS

Radical polymerization of acrylamide gels, even under carefully controlled conditions, rarely exceeds 90% conversion of the monomers into the polymer matrix. Thus, a 5%T gel will contain 30-60 mM unreacted free acrylamide. During isoelectric focusing (IEF), at pH > 8, free SH groups in proteins can rapidly react with the double bond of the free monomers in the gel. When Cys is incubated at room temperature with 30 mM acrylamide at pH 9.0, addition to the double bond is complete in 90 min. The existence of the Cys-acrylamide adduct has been ascertained by NMR, mass spectra and elemental analysis of the purified compound. It is suggested that acrylamide gels for IEF be treated with the same protocol adopted since 1980 for immobilized pH gradient gels: an "empty" matrix should be prepared, washed free of contaminants (unreacted monomers and catalysts, e.g., 3 × 1 h in excess distilled water) and stored dry for subsequent use. © 1990.