Immunological approach for the identification of isozymes of CAM-stimulated phosphatase.
Monoclonal antibodies are frequently used to identify protein isoforms. The present study documents certain artifacts in such applications and suggests methods for their testing. Two alpha subunit specific anti-calcineurin antibodies, VJ6 and VD3, reacted strongly with a brain isozyme, BPI, but not with the phosphatases from liver and spleen. Controlled proteolysis of BPI indicated that epitopes of both antibodies are localized to aminoterminal region, thus is suggesting that the lack of antibody reactivity could result from proteolytic artifacts. The occurrence of proteolysis during sample preparation can be monitored by including small quantity of BPI in the tissue extraction buffer. Rapid isolation procedure has been used to obtain liver and spleen isozymes reacting with VD3 but not VJ6 antibody.
Yokoyama
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Department of Medical Biochemistry, University of Calgary, Alberta, Canada.
Ali
Z
Z
Wang
J H
JH
D000818:Animals; D000911:Antibodies, Monoclonal; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D002148:Calmodulin-Binding Proteins / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D002417:Cattle; D000939:Epitopes / Q000032:analysis; D007527:Isoenzymes / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D008099:Liver / Q000201:enzymology*; D009928:Organ Specificity; D010449:Peptide Mapping; D010749:Phosphoprotein Phosphatases / Q000032:analysis* / Q000276:immunology / Q000302:isolation & purification; D013154:Spleen / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D002148:Calmodulin-Binding Proteins; D000939:Epitopes; D007527:Isoenzymes; D010749:Phosphoprotein Phosphatases