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Electrotitration curves of human gastric pepsinogens in agarose gels
Author(s) -
Majercakova Petra,
Kucerova Zdenka,
Desvaux F.Xavier,
Peltre Gabriel
Publication year - 2000
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/1522-2683(20000801)21:14<2919::aid-elps2919>3.0.co;2-h
Subject(s) - agarose , isoelectric focusing , pepsin , isoelectric point , chemistry , chromatography , immunostaining , pi , nitrocellulose , biochemistry , microbiology and biotechnology , biology , membrane , immunohistochemistry , enzyme , immunology
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 (p I ) 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 p I s, a simple and very fast 10 s pressure blot technique was applied. Human pepsinogens were separated alone or mixed with p I 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 p I values, and is suitable for studies of the pepsinogen polymorphism and its role in gastric diseases.