SAMPLE STREAKS AND SMEARS IN IMMOBILIZED PH GRADIENT GELS

In immobilized pH gradient (IPG) gel formulations as wide as pH 4-9, e ncompassing neutrality and containing the pK 7.0 acrylamido buffer as one of the buffering ions, smears are directly proportional to the tot al amount of the pK 7.0 species. At a total level of 10 mM pK 7.0 in t hese gel formulations, severe smears occur not only for mildly hydroph obic proteins (e.g., recombinant alcalase and termamylase) but also fo r the relatively hydrophilic pi marker proteins. Streaks and smears ar e essentially abolished in recipes devoid of the pK 7.0 compound or in formulations containing a maximum of 3 mM of this component. Although partitioning in water/n-octanol has shown the pK 7.0 acrylamido buffe r to be quite hydrophobic (P=0.5), the occurrence of smears could be t o the presence of oligomers in some commercial preparations. Even when dissolved in n-propanol, some batches of acrylamido buffers might sti ll contain oligomers, probably formed during the synthetic step.