Development of fast and simple chromogenic methods for glucan phosphatases in-gel activity assays.
Glucan phosphatases are essential for normal starch degradation in plants and glycogen metabolism in mammals. Here we develop two chromogenic methods for the detection of glucan phosphatase activity in situ after non denaturing poliacrylamide gel electrophoresis; one method uses pNPP and the second one applies BCIP/NBT. The assays are sensitive, fast, simple, reliable and cost-effective preventing the use of radioactive or fluorogenic compounds. Taking advantage of an efficient separation method combined with the reported assays it is possible to obtain information about oligomeric state of the active enzymes as well as to simultaneously detect glucan substrate binding and phosphatase activity.
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, 2000 Rosario, Santa Fe, Argentina.
Julieta B
JB
Carrillo
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, 2000 Rosario, Santa Fe, Argentina.
Diego F
DF
Gomez-Casati
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, 2000 Rosario, Santa Fe, Argentina. Electronic address: busi@cefobi-conicet.gov.ar.
Maria V
MV
Busi
Centro de Estudios Fotosintéticos y Bioquímicos (CEFOBI-CONICET), Universidad Nacional de Rosario, Suipacha 531, S2002LRK Rosario, 2000 Rosario, Santa Fe, Argentina. Electronic address: martin@cefobi-conicet.gov.ar.
Mariana
M
Martín
D017360:Arabidopsis / Q000201:enzymology*; D029681:Arabidopsis Proteins / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D006005:Phosphorylases / Q000737:chemistry*; D013194:Staining and Labeling / Q000379:methods*
D029681:Arabidopsis Proteins; D006005:Phosphorylases
Chromogenic substrate; Glucan phosphatase; In-gel assay; Native-PAGE