Electrotitration curves (ETC) of a marker
protein mixture, pH 2.5-5.65, and human
pepsinogens were performed in an
agarose gel, containing 2%
acid carrier ampholytes, forming a pH range of 2.5-5. Although the establishment of the pH gradient by isoelectric focusing was not quite complete and linear, both biochemically and immunochemically different types of
pepsinogen C (PGC) and
pepsinogen A (
PGA)
zymogens as well as the
acid isoelectric points (pI) marker
proteins were separated with good resolution. Three main fractions of
PGA (Pg3, Pg4, and Pg5) were detected. To obtain an exact determination of the
pepsinogen pIs, a simple and very fast 10 s pressure blot technique was applied. Human
pepsinogens were separated alone or mixed with pI marker
proteins in the pH range 2.4-5.65. No effect of the markers was observed on the
pepsinogen migration. To visualize the different
protein samples in the gel and on
nitrocellulose membrane, we have used
colloidal gold (
AuroDye) staining, proteolytic activity, and immunostaining with
monoclonal antibodies anti
PGA and PGC. The described method shows an ability to separate
proteins at acidic conditions with a resolution comparable to isoelectric focusing with immobilized pH gradients, but much faster, easier, and cheaper. In addition, the technique allows us to determine precise and exact pI values, and is suitable for studies of the
pepsinogen polymorphism and its role in
gastric diseases.