Sample-streaks and smears in immobilized pH gradient gels.
In immobilized pH gradient (IPG) gel formulations as wide as pH 4-9, encompassing neutrality and containing the pK 7.0 acrylamido buffer as one of the buffering ions, smears are directly proportional to the total amount of the pK 7.0 species. At a total level of 10 mM pK 7.0 in these gel formulations, severe smears occur not only for mildly hydrophobic proteins (e.g., recombinant alcalase and termamylase) but also for the relatively hydrophilic pI marker proteins. Streaks and smears are 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 buffer to be quite hydrophobic (P = 0.5), the occurrence of smears could be to the presence of oligomers in some commercial preparations. Even when dissolved in n-propanol, some batches of acrylamido buffers might still contain oligomers, probably formed during the synthetic step.
Esteve-Romero
J
J
Universitat Jaume I, Departament de Ciencies Experimentals, Area de Quimica Analitica, Castellò, Spain.
Simò-Alfonso
E
E
Bossi
A
A
Bresciani
F
F
Righetti
P G
PG
D005782:Gels; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000379:methods*; D013381:Subtilisins / Q000032:analysis
D005782:Gels; D013381:Subtilisins